PROTON ATPase ARE INVOLVED IN METASTASIS

IN HUMAN BREAST CANCER CELL

by

DEFENG LUG, M.S.. B.M.S.

A THESIS

IN

PHYSIOLOGY

Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center

in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Advisory Committee

Raul Martinez-Zaguilan (Chairperson) Szindor Gyorke

Narine Sarvazyan

Accepted

• v - . . y . r* ^ - ^ — ' - — • - ' - » » ^ »• — -»•.. ——

Associate Dean of the Graduate School of Biomedical Sciences Texas Tech University Health Sciences Center

December, 2001

PROTON ATPase ARE INVOLVED IN METASTASIS

IN HUMAN BREAST CANCER CELL

by

DEFENG LUO, M.S., B.M.S.

A THESIS

IN

PHYSIOLOGY

Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center

in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Advisory Committee

Raul Martinez-Zaguilan (Chairperson) Szmdor Gyorke

Narine Sarvazyan

Accepted

Associate Dean of the Graduate School of Biomedical Sciences Texas Tech University Health Sciences Center

December, 2001

ACKNOWLEDGMENTS C ^

I would like to take this opportunity to thank all the people who made this

endeavor possible. My father, Guangjin Luo, the first mentor in my life, who not only

gave me life but also encouragement, wisdom, and a hard working attitude, even though

he was sick when I was very young. I went to medical school partly because of the

motivation of curing him in order to pay back a little of what he gave to me, but he

passed away before I graduated fi-om the medical school. I owe him forever. My mother,

the greatest mother of the world, devoted all her love to me and my little brother and

sisters. She always supports me but whenever I ask if she needs help she always says: do

whatever you need to do; I can handle everything at home. Her attitude encourages me to

go further and further in life.

My brother, sisters and their spouses have always been by my side.

I can never thank enough my mentor, Dr. Raul Martinez-Zaguilan. It is he who

opened the door of the United States for me. Our relationship is like the taste of wine, the

longer I stay with him the more enjoyment I feel. He is supportive and suggestive when

problems arise, yet strict and demanding on the level he expects. His motivation and

energy never ebbed. His energy inspires everybody aroimd him and will continually

inspire me for the rest of my life.

I would like to express gratitude to my committee members (Drs. Gyorke and

Sarvazyan) for being patient with the completion of my Master of Science.

11

I am deeply in debt to the people in the lab, Gloria Martinez, Jose Rojas and

Shankar Sanka who taught me every single thing I know fi-om the lab. Without you guys

this work could not have been done.

The "Boys of Physiology," Joe, Shankar, Andy and Lee, I caimot imagine these

two and half years without you guys. Your support and fiiendship have meant the world

to me. I will always treasure your friendship.

To all my friends in the lab, Geraldine Tasby, my English teacher during the first

year, Hesham Attaya and Callenda Hacker, English teachers during the second year.

Huaxu Cheng, the only person I can talk to in my mother language in the lab and all those

who came and went, leaving me with good memories.

Special acknowledgement to Callenda for the proofireading of this thesis.

Special thanks to Dr. Orem and all the members in the physiology family, for

having me in such a wonderful family of physiologists. With you all I have had the best

two years of my Hfe.

Technical and Financial Support

I would like to acknowledge the technical support of Gloria Martinez, Karina

Bakunts, and Michelle Watts.

The work has been supported by grants to Dr. Raul Martinez-Zaguilan fi-om

American Cancer Society (RPG_00-35-01-CNE) and NIH (1 ROl H165695).

Ill

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii

ABSTRACT vi

LIST OF TABLES viii

LIST OF FIGURES ix

CHAPTER

L INTRODUCTION 1

Structure and Function of V-H^ATPase 1

V-H^ATPase and Tumorigenesis 7

V-H^ATPase and Tumor Chemotherapy 12

Rationale of Present Study 16

IL MATERL\LS AND METHODS 18

Cell Culture 18

Media, Buffer, and Chemicals 18

Fluorescence Spectroscopy 19

Spectral Imaging Microscopy 20

Laser Scanning Confocal Microscopy 23

Immunocytochemistry 26

Data Analysis 27

Invasion and Migration Assay 29

ATPase Assay 30

IV

Statistical Analysis 31

m. RESULTS 32

V-H^ATPases Colocalize with Actin in the Leading Edge of the Cancer Cells 32

Bafilomycin Ai Inhibits the Invasion of the Breast Cancer Cells 34

Proton Fluxes Are Larger in Highly Invasive Breast Cancer Cells 35

Intracellular pH Gradients Exist in Breast Cancer Cells and Are Larger in Highly Invasive Cells 38

Proton Pump Activities Are Elevated in the Leading Edge ofthe Breast Cancer Cells 40

Bafilomycin Sensitive ATPase Activities Were Discovered in the Plasma Membrane Fractions ofthe Breast Cancer Cells 40

rV. DISCUSSION AND CONCLUSION 44

V-H' ATPase and Tumor Invasive Behavior 44

Vacuolar H" ATPase and Tumor Metastasis 49

REFERENCES 52

* H i B

ABSTRACT

Cancer cells live in a more acidic environment than the normal tissue, but the

intracellular pH (pHin) of tumors is more alkaline than in non-tumor cells. This indicates

that cancer cells maintain a larger transmembrane pH gradient than normal cells. Our

previous studies suggested that vacuolar type H'* -ATPases (V-H'*'ATPases) that normally

reside in acidic organelles may be also expressed at the plasma membrane (pmV-

ATPase) of highly metastatic human tumors. We hypothesize that pmV-ATPase plays a

role in maintaining an alkaline intracellular environment favorable for growth, while

maintaining an acidic extracellular environment favorable for invasion. Human breast

cancer cell lines with distinct metastatic potential (MCF-7, MB-231, MB468 and MB-

435s) were used as experimental models. Immunocytochemical experiments showed that

pmV-ATPases are located at the leading edge of metastatic human breast cancer cells.

We employed spectral imaging and line scanning confocal microscopy (LSCM) to

monitor the pHfn of discrete cellular regions, as well as fluorescence spectroscopy to

determine proton fluxes in these cancer cell lines. These approaches are complementary

in that they offer unsurpassed spatial, temporal and spectral resolution. Our data show

that the pHin is more alkaline at the leading than at the lagging edge in the more invasive

cells. The magnitude ofthe pHin gradient is larger in the highly than in the lowly

metastatic cells. The proton fluxes are faster in the highly than in the lowly metastatic

cells. Pharmacological and ion substitution experiments indicated that pmV-H"^ATPase

expression was more elevated in the highly than in lowly metastatic cells. Invasion assays

of these cancer cells lines show that bafilomycin Ai inhibits the invasive behavior. These

VI

novel observations suggest that the magnitude ofthe pH gradients is determined by pmV-

H^ ATPase, and pmV-H^ ATPase is involved in the invasion ofthe cancer cells.

Vll

LIST OF TABLES

Subunit composition of V-H^ATPases 3

Results of invasion and migration 35

Vlll

^ • • ~ ' " * - ^ -•r.T

LIST OF FIGURES

1 Structure of V-H""ATPase 4

2 Schematic illustration of the spectral imaging system 22

3 The procedure of spectra generation 24

4 Line scan confocal microscopy (LSCM) reveals intracellular

pH gradients 25

5 V-H'^ATPases are shown on the cell surface 33

6 Bafilomycin Ai inhibits invasion of the breast cancer cells 36

7 Highly invasive cells exhibit larger proton fluxes than lowly invasive cells 37

8 Highly invasive cells exhibit larger pH gradients 39

9 Spectral imaging microscope shows intracellular pH gradients exist fi-om leading to lagging edge ofthe MDA-MB231 breast cancer cells 42

10 ATPase assays show bafilomycin Ai sensitive ATPase activities 43

IX

CHAPTER I

INTRODUCTION

The vacuolar or plasma membrane H*-ATPases (V-H"ATPase or pmV-

H^ATPases) are members of a family of ATP-driven proton pumps responsible for the

proton concentration regulation ofthe intracellular compartments and the cytoplasm. It is

one ofthe most fundamental enzymes in nature. It functions in almost every eukaryotic

cell and energizes a wide variety of organelles and membranes (Forgac 1998, Forgac et

al. 1999, Stevens et al. 1997, Nelson et al. 1999, Kane 1999, Putnam 1997).

Proton-ATPase was related to cancer since late 80s of last century, when two

Spanish scientists expressed yeast plasma membrane proton pump in the fibroblasts and

tumorigcnizcd the cells (Perona et al. 1988). V-H^ATPase was found to be functionally

expressed in the plasma membrane of human tumor cells (Martinez-Zaguildn et al. 1993).

In this chapter, we try to summarize the newest progress in this area. The roles of V-

H^ATPasc in tumorigenesis and drug resistance are discussed as well as the structure and

function of V-H^ATPase as background for this relatively new area of study.

Structure and Function of V-H" ATPase

From sequence homology and overall structure comparison we know that V-

H""ATPase is related to the F-ATPases (Arai et al. 1998, Zimniak et al. 1988, Adachi et

al. 1990, Bowman et al. 1988), which are involved in ATP synthesis in mitochondria,

chloroplasts and bacteria (Weber et al. 1997, Fillingame et al. 1997, Cross et al. 1996).

Similar to Fi and Fo in the F-ATPase, the V-H^ATPases are composed of two ftinctional

domains (Forgac 1998, Forgac et al. 1999, Stevens et al. 1997, Putnam 1997). The Vi

domain is a 570-kDa peripheral complex responsible for hydrolysis of ATP. Vi is

composed of eight different subunits, named A-H, with three copies each ofthe A and B

subunits and single copies ofthe remaining subunits (Arai et al. 1988) (Table 1, Figure

1). The A and B subunits both participate in nucleotide binding, with the catalytic site

located on the A subunit (Forgac 1998, Stevens et al. 1997, Nelson et al. 1999). The VQ

domain is a 260-kDa integral complex that is responsible for proton translocation across

the membrane. Fo contains five different subunits (subunits a, d, c, c', and c"), with six

copies ofthe c/c' subunits and single copies ofthe other subunits (Arai et al. 1988).

Energy coupling between Viand Vo is thought to occur through the physical rotation of a

stalk that connects the two parts ofthe enzyme complex. This mechanism implies that

beside the center stalk, astator-like structure is needed to prevent the rotation ofthe

headpiece relative to the membrane-bound part. Both center stalk and additional stator-

like stalk were found in the V-type Na -ATPase of Clostridium fervidus as determined by

electron microscopy (Boekema et al. 1997). A similar finding was reported for the F-

ATPase of Escherichia coli (Liu et al. 1996, Abrahams et al. 1994). Negatively stained

images ofthe bovine-coated vesicle V-H^ATPase reveal an even more extensive

secondary stalk (Forgac 1998). This peripheral stalk is crucial for the current rotary

model of ATP-driven proton transport by both the F- and V-H^ATPases (Cross et al.

1996,Viketal. 1994).

Table 1. Subunit composition of V-H^ATPases

Domain

V,

Vo

Subunit

A B C D E F G H a

d c c' c"

Molecular mass

in Bovine

73 58 40 34 33 14 15 50

100

38 17 17 19

in Yeast

69 57 42 32 27 14 13 54 95

36 17 17 23

Subunit function

Catalytic site, regulation (?) Noncatalytic site, targeting (?) Activity, assembly Activity, assembly Activity, assembly Activity, assembly Activity, assembly Activity (not assembly) H+ transport, assembly, targeting Binding site of bafilomycin Al Activity, assembly H+ translocation, DCCD site H+ translocation, DCCD site (?) H+ translocation

Adapted and modified from Forgac, M in J Boil Chem 1999; 274:12951-12954

The roles of other subunits (subunits C-H) in Vi domain are primarily to assemble

the V-H^ATPase complex. Subunit D fiinctions as shaft and subunit E functions as the

hook (Nelson et al. 1999, Tomashek et al. 1996). The other subunits are not clearly

documented yet. Ofthe five different types of subunits in V, domain, three (c, c', and c")

are highly hydrophobic proteins, termed proteolipids. These three proteolipids were

recently characterized as V-H+-ATPase proton pore in yeast (Powell et al. 2000). They

are homologous both to each other (Hirata et al. 1997) and to the c subunit ofthe F-

ATPases (Fillingame 1997). The F-ATPase c subunit is composed of two transmembrane

helices (TMjand TM2) containing a single buried carboxyl group in TMj that is essential

Proton Pump

A

l l l t l tMf i l ) ) \..x'^

i) ' ' i ' 1 ^ •

/ i

ff ^ ^ ^ 1

c

c

c a U » 111111 M l |

%

I ( f f l f i ( f i § t C < f f f C K H l f f t

- ^ D

ATP

Figure 1. Structure of V-H^ATPase

for proton transport (Fillingame 1997). The V-H^ATPase subunits c and c' are composed

of four transmembrane helices containing a single essential carboxyl group in TM4

(Hirata et al. 1997). Both c and c' ofthe V-H"ATPases were derived by gene duplication

and fusion fi-om the F-ATPase c subunit (Nelson 1992). Because there are 12 copies of

subunit c in Fo (Fillingame 1997) and 6 copies of subunits c/c* in Vo (Arai et al. 1988),

these subunits contribute the same number of transmembrane helices (24) to their

respective domains. The number of buried carboxyl groups contributed by the c subunits

in Vo, however, is only half that in FQ and this has been suggested to account for the

difference in H^ /ATP stoichiometry observed for the V- and F-ATPases (Cross et al.

1990). For both Vo and Fo, reaction of a single c subunit with dicyclohexylcarbodiimide

(DCCD), which covalcntly modifies the carboxyl side-chain specifically, is sufficient to

completely block proton transport (Forgac 1998, Fillingame 1997). This indicates that all

the buried carboxyl groups must be functional for proton transport to occur. By contrast

with subunits c and c', subunit c" is a 23-kDa protein that contains five transmembrane

helices, with the essential carboxyl group in the third transmembrane segment (Hirata et

al. 1997). Subunit a (lOOkDa) ofthe V-H^ATPase is a transmembrane glycoprotein

possessing an N-terminal hydrophilic domain and a C-terminal hydrophobic domain

containing multiple putative transmembrane helices (Perin et al. 1991). It maybe involved

in targeting the V-H"* ATPase in different compartments ofthe cell (Goldstein et al.

1991). Evidences obtained suggest that the 100-kDa subunit possesses the binding site for

the highly specific V-H^ATPase inhibitor bafilomycin (Zhang et al. 1994). More

recently, three isoforms ofthe 100-kDa subunit were cloned (Nishi et al. 2000). By

comparing the reversible disassembling ability of F- and V-type ATPases, Kane found

that a ftindamental difference is the stability ofthe F, Fo and V, Vo complexes in the cell.

The F, and Fo sectors associate to form a stable complex in the cell and are rarely, if ever,

present in their dissociated form. In contrast, dissociated V, and Vo sectors can be simply

induced by glucose deprivation. It suggests that the ftinction of V-H^ATPase can be

regulated by the extracellular conditions (Kane 1995, 2000).

As die name indicates, vacuolar or plasma membrane H^ -ATPases are

responsible for proton translocation which controls the acidification of intracellular

compartments and also ofthe regulation ofthe pH^ in eukaryotic cells (Martinez-Zaguil^

et al. 1993, Forgac 1998, Stevens et al. 1997, Nelson et al. 1999). Acidification of

vacuolar compartments plays an important role in a variety of cellular processes,

particulariy membrane traffic processes. In receptor-mediated endocytosis, acidification

of endosomes serves as a signal that activates release of internalized ligands (such as, low

density lipoprotein and insulin) from their receptors. This uncoupling allows unoccupied

receptors to recycle to the cell surface where they can be reutilized, whereas the ligands

are targeted to lysosomes for degradation (Stevens et al. 1997). Acidification of

endosomes is also required for the formation of endosomal carrier vesicles, which are

involved in moving ligands along the endocytic pathway (Clague et al. 1992), and in

activating fusion between the endosomal membrane and internalized envelope viruses

(such as influenza virus) that is necessary for viral infection (White et al. 1992).

In addition to their role in intracellular compartments, V-H"^ATPases have also

been shown to play an important role in the plasma membrane of various specialized

cells. Thus V-H""ATPases in the apical membrane of renal intercalated cells ftinction in

renal acidification (Gluck 1992), whereas plasma membrane V-H^ATPases in

macrophages and neutrophils assist in pH homeostasis (Swallow et al. 1993). V-

H ATPases in the midgut of insects have been shown to establish a membrane potential

across the apical membrane that drives K+ transport via an electrogenic H"' /K^ antiporter

(Wieczorek et al. 1991). Using the specific V-H^ATPase inhibitor bafilomycin Ai

(Bowman 1988), V-H^ATPases have been implicated in apoptosis (Gottlieb 1995). V-

H" ATPases can be targeted to the plasma membrane in osteoclasts (Chatterjee et al.

1992) and tumor cells (Martinez-Zaguilan et al. 1993) where they create an acidic

extracellular environment that is necessary for bone resorption and metastasis,

respectively. The alkaline pHjn is favorable for cell growth and proliferation while an

acidic extracellular environment is preferable for tumor invasion (Martinez-Zaguilan et

al. 1996) and metastasis (Martinez-Zaguilan et al. 1998) (more detail in later).

V-H^ATPase and Tumorigenesis

Macroscopic measurements of pH have shown that the medium surrounding the

solid tumors is generally in the acidic range (Griffiths 1991). However, the cytosolic pH

ofthe tumor cells is generally alkaline (Stubbs 1995, Martinez-Zaguilan et al. 1993).

Combining these two fundamental discoveries, we can easily conclude that tumor cells

must be able to extrude protons more efficiently than their normal counterparts. On the

other hand, a common early response of eukaryotic cells to stimuli that activate their

proliferation is an increase in pHjn (Nuccitelly 1982). Subsequent experiments by our

group indicated that pH played a permissive role for cell proliferation, i.e., changes in pH

were necessary but not sufficient for the proliferative response (Gillies et al. 1991). These

experiments, however, did not address the role for changes in pH in tumorigenesis.

To more direcUy address the question ofthe role of pH in cell transformation,

Perona and Serrano transfected fibroblasts with the gene encoding for the yeast pmV-

H ATPase. This resulted an elevated pHjn and the acquisition ofthe tumorigenic

phenotype in transfected cells. These cells are serum-independent for growth, clone in

soft agar, and are tumorigenic when injected into nude mice (Perona et al. 1988, 1990,

Gillies et al. 1991, Peterson et al. 1994). These eariier experiments, however, did not

address the question of whether their pHjn was elevated under physiological conditions,

i.e., bicarbonate-containing media. This is essential to understanding the role of pHjn in

cell tumorigenesis, since bicarbonate in the media may have had altered the interpretation

ofthe data. To further address this issue, a series of experiments were done in our

laboratory.

By transfecting yeast H -ATPase into fibroblast cells, which rendered them

tumorigenic, we found that these transfected cells do maintain a higher pH ^ than control

cells. Paradoxically, the transfected cells did not grow faster than their normal

counterpart under normal culture conditions but they did grow to a much higher density

when they were cultured in serum fi-ee medium. These transfected cells glycolyzed much

more rapidly than phenotypically normal cells, which raised the possibility that

supraphysiological pHin circumvents other mitogen-induced events and stimulates growth

8

directly (Gillies et al. 1990). These data suggested that elevated pH.n is possibly a

proliferative trigger in situ.

While both H^ATPase transfected and non-transfected cell lines showed similar

growth increases in response to platelet-derived growth factor (PDGF)-BB and epidermal

growth factor (EGF), they responded differently to insulin, insulin-like growth factor-I

(IGF-I) and PDGF-AA. H^ATPase transfected cells (RNla line) exhibited increased

growth at nanomolar concentrations of insulin but the parental cells had only a relatively

minor response to insulin at 10 ^M. The two cell lines also responded differentiy to

PDGF-AA. The transfected cells were relatively insensitive to stimulation by PDGF-AA

and express fewer PDGF alpha receptors as shown by Northern blots and receptor-

binding studies. Differences in insulin and IGF-I receptor number alone could not explain

these results (Peterson et al. 1994). These results suggested that the H^-ATPase activated

a downstream element in the PDGF-AA signal transduction pathway that complements

insulin and IGF-I signals, while leading to down-regulation ofthe PDGF alpha receptor.

The protein turnover rate ofthe transfected cells and a low-activity mutant H"*-

ATPase transfected cell line (N-Mut line) were examined (Gunn et al. 1994). The protein

degradation rates in the transfected cells were significanUy less than those measured in

NIH 3T3 and N-Mut cells. Rates of protein synthesis were the same for sparse cultures of

RNla and NIH 3T3 cells. However, as cell density increased, the protein synthesis level

declined in NIH 3T3 cells and remained high in RNla cells. These data indicate that

transformation with the H"* -ATPase has a direct effect on rates of protein degradation,

possibly through an elevation of pH. The higher pHvac will directly affect lysosomal

protein breakdown and the higher pHcyt may be permissive for maintenance of low basal

rates of protein breakdown. Altogether, it suggests that transformation with the H -

ATPase provides a permissive environment for high rates of protein synthesis and low

rates of protein degradation that result in high rates of growth and the tumor phenotype

(Gunn et al. 1994). The other metabolic properties of these transfected cells were also

investigated. Higher rates of glycolysis were observed in the transfected cells because of

the presence of low affinity glucose transporters (Martinez et al. 1994). The maximal

velocity (V^ax) for glucose utilization was up to sixfold higher in RNla cells than in the

NIH 3T3 cells, suggesting that the number of glucose transporters is higher in RNla than

NIH 3T3 cells. Glucose addition to NIH 3T3 cells resulted in modest decreases in both

pHj,, and [Ca ]j„. In contrast, RNla cells responded to glucose with a large decrease in

pHjn followed by a large decrease in [Ca ^ Jj . This suggests that expression of

plasmalemmal H -ATPase render the cells tumorigenic by affecting a number of

processes, including pHj , [Ca ]j„ and glucose transport.

These experiments with transfected yeast- H -ATPase were important in that they

allowed us to conclude that overexpression of a H -ATPase at the plasma membrane was

important for the acquisition ofthe transformed phenotype. However, they did not

address the issue of whether human tumor cells express H -ATPase at the plasma

membrane, which may explain the acquisition ofthe transformed phenotype. These

earlier experiments did not allow us either to understand the relevance of H" -ATPase in

multistage carcinogensis. Nevertheless, these earlier findings were critical in establishing

our rationale that overexpression of H -ATPase was sufficient to induce a transformed

10

phenotype. These data also indicated that overexpression of H -ATPase at the cell

surface was the primary event leading to a transformed and tumorigenic phenotype.

All the evidence fi-om the yeast H -ATPase transfected cell model suggested that

the expression of extra proton pumps on the normal cell membrane can increase the pHin

and the protein synthesis, decrease the protein degradation, and promote the cell growth.

These events trigger cell proliferation and finally tumorigenesis. However, yeast H" -

ATPase transfected cells are generally over expressed with the enzyme. The insertion of

H^-ATPase into the plasma membrane may induce effects other than increasing the

magnitude ofthe pH gradient. To elucidate the role that H""-ATPase may play in

tumorigenesis, evaluating the expression level of this enzyme in the natural occurring

cancer cell will be more convincible. V-H^ATPases do present in some tumor cells. In

the previous study of our lab, we screened 19 normal and human tumor cell lines for the

presence of plasmalemmal vacuolar type H -ATPase (pmV-H^ATPase) activity using

bafilomycin Aj to inhibit V-H+ATPase and SCH-28080 to inhibit P-type H -K+-ATPase.

Bafilomycin A] decreased pHjn in the six tumor cell lines with the highest resting pHjn in

the absence of HCO3'. SCH-28080 did not affect pHjn in any ofthe human cells.

Simultaneous measurement of pH in the cytoplasm and in the endosomes/lysosomes

localized the activity of bafilomycin to the plasma membrane in three cell lines. In the

second phase of this study, these three cell lines were shown to recover from NH4" -

induced acid loads in the absence of Na" . This recovery was inhibited by N-

ethylmaleimide, bafilomycin A], and ATP depletion, and was not significantiy affected

by vanadate, SCH-28080, or hexamethyl amiloride. All these results strongly suggested

11

that a V-H*ATPase was expressed in the plasma membrane of some tumor cells

(Martinez-Zaguilan et al. 1993). Combining the transfected cell model findings, we

concluded that V-H^ATPase may play a role in tumorigenesis.

Some other earlier evidence also suggested that V-H^ATPase might be involved

in carcinogenesis. Active electrogenic proton pump activation was found in Ehrlich

ascites tumor cells (Heinz et al. 1981). A series of tumorigenic Chinese hamster embryo

fibroblast (CHEF) cell lines were found to maintain a pHin that is 0.12 ± 0.04 pH units

above that ofthe nontumorigenic CHEF/18 parental line. Enhanced H+-extruding activity

was expressed upon transformation of these CHEF cells (Ober et al. 1987). ATP was

directly required for pHj regulation in both A-431 tumor cells (Cassel et al. 1986) and

EAT cells (Gillies et al. 1982). V-H^ATPases have been observed in the plasma

membrane of EAT cells, osteoclasts and macrophages (Blair et al. 1989, Swallow et al.

1990). These results suggested that V-H^ATPase maybe involved in the machinery

maintaining the cell proliferation (Manabe et al. 1993).

V-H""ATPase and Tumor Chemotherapy

A major obstacle for the effective treatment of cancer is the phenomenon of

multidrug resistance (MDR) exhibited by many tumor cells (Juranka et al 1989, Roninson

1987). Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp),

a drug efflux pump (Gottesman et al. 1993). However, most mechanisms of MDR are

complex, employing P-gp in combination with other ill-defined activities. Altered pHin

has been implicated to play a role in drug resistance (Altenberg et al. 1993, Keizer et al.

12

1989, Boscoboinik 1990). Decreased vacuolar acidification capacity has also been related

to drug resistance in rat liver preneoplastic nodules (Andersson 1989). The possibility of

the involvement of V-H^ATPase in multidrug resistance was examined in early 1990's.

HL60 cells isolated for resistance to vincristine (HL60/Vinc cells) or doxorubicin

(HL60/Adr cells) exhibited levels of an energy-dependent drug efflux pump (Marsh et al.

1986, McGrath et al. 1987). HL60/Vinc cells contained the drug transporter P-

glycoprotein, whereas the HL60/Adr isolate did not. Using bafilomycin Ai inhibited V-

H+ATPase activity at low concentrations. The results showed that bafilomycin Ai

induced a major increase in drug accumulation and inhibited drug efflux in both

HL60/Adr cells and HL60/Vinc cells. Similar results were obtained with 7-chloro-4-

nitrobenz-2-oxa 1,3 diazole, an agent that is also capable of inhibiting V-H^ATPase.

Azide, an inhibitor of Fi Fo mitochondrial ATPase, and vanadate and ouabain, which are

inhibitors of EiE2-type ATPase, did not affect drug levels in resistant cells. Bafilomycin

AI did not compete with [ H] azidopine binding to P-glycoprotein(Marquardt et al.

1991). Thus, bafilomycin A| does not appear to ftinction as a substrate for P-

glycoprotein. These results suggested an involvement of V-H^ATPase activity in the

pathway of drug efflux from HL60/Adr cells and HL60/Vinc cells.

A series of work has been done in our laboratory to investigate the mechanisms of

pHin regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells.

Ofthe drug-resistant lines, MCF-7/D0X (also referred to as MCF-7/D40) contained P-gp

and MCF-7/MITOX did not. No difference ofthe expression level of both NaV H+

exchanger and anion exchanger (AE) was found in these cell lines. The presence of pmV-

13

ATPase activity in these cell lines was then investigated. In the absence of Na and

HCO3", MCF-7/S cells did not recover from acid loads, whereas MCF-7/MIT0X and

MCF-7/DOX cells did. Furthermore, recovery of pHjn was inhibited by bafilomycin Ai

and NBD-Cl, potent V-H" ATPase inhibitors. The release of endosomally-trapped dextran

was more rapid in the drug-resistant compared with the drug-sensitive cells. Furthermore,

the drug-resistant cells entrapped doxorubicin in intracellular vesicles whereas the drug-

sensitive cells did not. One possibility to explain these results is that the measured pmV-

H"* ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane

turnover (Martinez-Zaguilan et al. 1999). The effect of acidic vesicle tumover on drug

resistance was investigated in the other studies both experimentally and theoretically. The

data indicated that tumover of acidic vesicles can be an important contributor to the drug-

resistant phenotype. The effectiveness of such a drug export mechanism was thought

comparable to drug extrusion via drug pumps such as P-gp, especially in cells that do not

overexpress plasma membrane-bound drug pumps like P-glycoprotein (Raghunand et al.

1999). After studying the mechanisms contributing to reduced cytotoxic drug

accumulation in two MDR human lung cancer cell lines without P-gp expression.

Versantvoort et al. (1992) concluded that the accumulation of drugs in the non-P-gp

MDR human lung carcinoma cell lines SW-1573/2R120 and GLC4/ADR is reduced by

an energy-dependent drug export mechanism that prevents efficient transport of drug to

the target.

Bafilomycin Ai, the V-H+ATPase specific inhibitor, was found to reversibly

disturb the fusion between autophagosomes and lysosomes. It also prevented the

14

appearance of endocytosed HRP in autophagic vacuoles. These results suggested that

acidification ofthe lumenal space of autophagosomes or lysosomes by V-H^ATPase was

important for the ftision between autophagosomes and lysosomes (Yamamoto et al.

1998). The intracellular vesicular compartments in tumor cells (MCF-7) were reported to

fail to acidify. This failure resulted in a significant decrease in the pH gradient (0.9 pH

unit) between the vesicular luminal compartments and the cytoplasm. These defects were

correlated with a disruption in the organization and function ofthe trans-Golgi network

(TGN) and the pericentriolar recycling compartment (PRC). In marked distinction, drug-

resistant tumor cells (MCF-7adr) derived from the MCF-7 line that were resistant to the

most widely employed chemotherapeutic drug, adriamycin, appeared normal in both

acidification and organization ofthe PRC and TGN. Treatment of drug-resistant MCF-

7adr cells with nigericin and monensin, ionophores demonstrated to disrupt vesicular

acidification (Tartakoff 1983), led to a resensitization of these cells to adriamycin. Drug

sensitivity was proposed to result from an acidification defect within vesicles ofthe

recycling and secretory pathways. A functional consequence of this defect was the

diminished capacity of cells to remove cytotoxic drugs from the cytoplasm by

sequestering of protonated drugs within the vesicles, followed by drug secretion through

the activity ofthe secretory and recycling pathways (Schindler et al. 1996).

In conclusion, V-H^ ATPases not only take part in the pHin regulation, but are also

involved in many aspects ofthe property ofthe cancer cells. The increased activity of

pmV ATPase maybe related to the invasive and metastatic phenotype ofthe human tumor

15

cells. This enzyme is also closely involved in the acquisition ofthe MDR phenotype of

the cancer cell.

Rationale of Present Studv

Breast cancer is the most common malignancy in women in the United States. It

accounts for approximately one third ofthe female malignancies (Parker SL et al. 1997).

Recent statistical data predicted that I in 8 women in the United States will develop

breast cancer during her lifetime (Ries LAG et al. 1998). What role does V-H""ATPase

play in this type of cancer? Answering this question may provide fiirther understanding

of characteristics of this cancer and consequently find a way to prevent or reduce the drug

resistance during chemotherapy. Cancer cells live in a more acidic environment than the

normal tissue, but the pHjn of tumors is more alkaline than non-tumor cells. This indicates

that cancer cells maintain a larger transmembrane pH gradient than normal cells. Our

previous studies suggested that V-H"* ATPases that normally reside in acidic organelles

might be also expressed at the plasma membrane (i.e., pmV-H^ATPase) of highly

metastatic human tumors.

We hypothesize that pmV-H" ATPase plays a role in maintaining an alkaline

intracellular environment favorable for growth, while maintaining an acidic extracellular

environment favorable for invasion. Human breast cancer cell lines with distinct

metastatic potential (MCF-7, MDA-MB-231, MDA-MB-468 and MB-435s) were used as

experimental models. In this project, immunocytochemistry ofthe V-H" ATPase and F-

actin were used to show the location of V-H"^ATPases in the metastatic human breast

16

cancer cells. Spectral imaging and laser scan confocal microscopy (LSCM) were

employed to monitor the pHjn of discrete cellular regions, as well as fluorescence

spectroscopy to determine proton fluxes in these cancer cell lines. Our data showed that

the pHjn was more alkaline at the leading than at the lagging edge in the more invasive

cells. The magnitude ofthe pHm gradient was larger in the highly than in the lowly

metastatic cells. The proton fluxes were faster in the highly than in the lowly metastatic

cells. ATPase assays, pharmacological and ion substitution experiments indicated that V-

H^ATPase expression was more elevated in highly than in lowly metastatic cells.

Invasion assay of these cancer cells lines showed that bafilomycin inhibited the invasive

behavior.

17

CHAPTER II

MATERIALS AND METHODS

Cell Culture

Human breast cancer cell lines, MDA-MB-231 (ATCC number HTB-26), MDA-

MB-468 (HTB-132), MDA-MB-435S (HTB-129) and MCF-7 (HTB-22), were purchased

from ATCC (American Type Culture Collection) and plated in culture dishes and grown

in DMEM (ICN Biomedical, Inc. Costa Mesa, CA) containing 2 mM glutamine, 24 mM

NaHCOj, 5 mM Glucose, supplemented with 10% FBS (Gibco, Grand Island, N.Y.)

under a 95% air-5% CO2 humidified environment at 37° C. For fluorescence

spectroscopy studies of pHjn with SNARF-1 in cell population, the cells were inoculated

onto 60 mm petri dishes containing 6 glass cover slips (20x8mm) coated with 1.5%

gelatin at densities of 1 x lO' cells/dish in the above noted medium until the cells reached

confluency. Cells were plated on 18 mm coverslips for immunocytochemistry and on 25

mm round coverslips for confocal and spectral imaging microscopy. For ATPase assays,

cells were plated to 100 mm petri dishes direcUy. Cells used for experiments were from

passages 5-20.

Media, Buffers, and Chemicals

Cell Suspension Buffer (CSB) contained: 1.3 mM CaCh, 1 mM MgS04, 5.4 mM

KCl, 0.44 mM KH2PO4,110 mM NaCl, 0.35 mM NaH2P04, 5 mM Glucose, 2 mM

Glutamine and 20 mM HEPES, at a pH of 7.4 at 37° C. Media pH was direcUy measured

18

with a Beckman Model 71 pH meter, using a Coming glass combination electrode. The

electrode was calibrated at two known pH values using commercially prepared standards

from VWR Scientific (San Francisco, CA.). In vitro calibrations were carried out using

the free acid forms of SNARF-1 solubilized in High K^ pH 5.5. SNARF-1 (1 mM stock)

free acids were dissolved in dimethyl sulfoxide and maintained at -80°C in the dark until

used.

Na -free CSB consisted of all CSB ingredients, except those containing sodium.

110 mM N-methyl-glucamine was substituted for NaCl. High K" buffer contained 146

mM KCl, 20 mM NaCl, 5 mM glucose, 2 mM glutamine, 10 mM HEPES, 10 mM MES,

and 10 mM bicine. The rationale for using these organic buffers was to allow for precise

buffering across a wide pH ranging from 5.0-9.0 (Martinez-Zaguildn et al, 1996).

Bafilomycin Ai was obtained from Wako Chemicals (Richmond, VA). The

fluorescent dyes were obtained from Molecular Probes (Eugene, OR). All other

chemicals were obtained from Sigma Chemical (St. Louis, MO), unless otherwise stated.

Fluorescence Spectroscopy

Intracellular pH was determined by the fluorescence of SNARF-1 (5-[and-6]

carboxy-SNARF-1) as described previously (Martinez-Zaguilan et al, 1991). The dye is

membrane permeable in the acetoxymethylester (AM) form. Once in the cell the dye is

cleaved by cell esterases to produce the free acid form ofthe dye, which is membrane

impermeable. Two cover slips containing cells at confluency were placed into a 60 mm

petri dish with 3.0 ml CSB (pHex 7.4) and l\iM SNARF-1-AM and incubated at 37EC in

19

5% C02 for 45 minutes on a rocker platform (Cole-Parmer, Vernon Hills, IL). The cells

were then rinsed with excess CSB, and ftirther incubated at the pH being studied for 30

minutes at 37EC in 5% CO2 to ensure complete ester hydrolysis and leakage of uncleaved

dye. The two cover slips were placed back-to-back in a holder perfusion device and

perfused at a rate of 3 ml/min and the fluorescence of SNARF-1 was monitored with an

SLM-8100/DMX spectrofluorometer (Spectronics Instruments, Rochester, NY) equipped

for sample perfusion. The sample temperature was maintained at 37EC by keeping both

the water jacket and perfusion media at 37EC using an iso-temperature immersion

circulator water bath (Lauda model RM20, Brinkmann Instruments, Westbury, NY). All

measurements were performed using 4 nm-bandpass slits and an external rhodamine

standard as a reference. Fluorescence was monitored in continuous acquisition mode by

using an excitation wavelength of 534 nm and monitoring emissions at 584, 600, 644 nm

as described elsewhere (Martinez-Zaguilan et al, 1991). The fluorescence emission at

584 nm decreases and that at 644 nm increases with increasing pH; as such the ratio of

644/584 was used to monitor pH changes. The 600 nm wavelength, which is insensitive

to pH, was used to evaluate the efficiency of dye loading, quenching or other artifacts.

Fluorescence data were converted to ASCII format for analyses.

Spectral Ima^ng Microscope

Figure 2 shows the schematic representation ofthe major components required for

Spectral Imaging Microscopy (SIM). Briefly, an OLYMPUS IX 70 inverted microscope

was equipped with a 100 Watt Hg lamp as an illumination source. Imaging optics

20

included a 60X 1.4 NA OLYMPUS objective and a 5.4X eyepiece to focus the cell image

onto the input slit of a grating monochromator (Aries 250IS/SM spectrograph, Chromex,

Inc., Albuquerque, NM). A grating blazed at 300 lines/inch provided a spectral

bandwidth of 200 nm. The spectral output from the grating was imaged onto a liquid

cooled CCD camera (Photometries Mod. CH350, Tucson, AZ) equipped with a 512 x 512

element imaging chip that is 60% quantum efficient at 546 nm (Techtronics, Tucson,

AZ). The output image was composed of spectra acquired at multiple positions along the

length ofthe entrance slit. The spectral image occupied less than VA ofthe serial register

ofthe imaging chip and for a single cell image it occupies significantly less area.

Therefore, internal shifting of data allowed for rapid sequential image acquisition.

Adequate signal could be obtained with as little as 2 msec exposure time. Read out ofthe

full chip required 500 msec. Spectral resolution was primarily dependent on the grating

resolution, but also on the width ofthe entrance slit. As slit width was increased, signal

increases substantially while spectral resolution suffers comparably less (Martinez-

Zaguildn et al. 1996). Thus, spectral resolution was sacrificed for improved signal-to-

noise ratio. In the current experiments the entrance slit was set at 200 fim. Mapping ofthe

wavelength to position on the CCD chip was performed by reflecting light from the Hg

lamp to the monochromator. The grating was scanned to position the 610 line ofthe Hg

lamp near one end ofthe output spectrum and the doublet peak at 584/644 imaged near

the center. The digital output ofthe CCD camera was stored in a PC with 128 MB RAM.

Image analysis was done on a PC 300 MHz /128MB RAM using MAPS analysis

21

Eyepiece Spectrograph

Figure 2. Schematic illustration ofthe spectral imaging microscope system. The light source is alOO Watt mercury lamp(Hg Lamp). The specific excitation ofthe fluorophore is selected using anexcitation filter (EX. Filter). The emissions passes through an

adjustable entrance slit and be collected by the spectrograph with an attached Charge Coupled Device (CCD) camera, which connected to a computer. Using software MAPS-2 the computer give out the spectra ofthe emissions captured.

22

software version 2.0 (Photometries, Tucson, AZ) and further analyzed using MS Excel

2000 (Microsoft Co., Redmond, WA) and Sigma Plot for Windows, version 5.0 (Jandel

Scientific, San Rafael, CA) (Figure 3).

Subconfluent cultures of breast cancer cells were grown on 25 mm round glass

coverslips loaded with 7 fiM SNARF-l/AM as mentioned in fluorescence spectroscopy

experiment. Then the coverslips were mounted onto a special chamber and placed in a

special temperature controlled perfiasion stage PDMI-2 (Medical Systems Inc.,

Greenvale, NY), with a buffer perfusion rate of 3 ml/min. This was placed on the sample

stage of 1X70 Olympus inverted microscope.

Laser Scanning Confocal Microscope

Confocal Microscopy was performed with a Bio-Rad MRC1024 confocal

microscope (Rojas et al. 2000). Subconfluent breast cancer cells were loaded with 7 |iM

SNARF-l/AM and mounted to the same chamber and temperature controlled perfusion

stage as described above for spectral imaging microscope. A single cell was visualized in

each coverslip. Line scans were carried out by drawing a line across the cell from the

leading edge to the lagging edge. Four regions of interest were selected on the line for

each cell. Using 514 nm argon laser for excitation, the emissions were collected at 584

nm and 644nm from the interest regions every 2 milli-seconds during scanning (Figure

4). The data was acquired on an 0S2 based computer using tiie Lasersharp software.

Intracellular pHs were calculated with the ratio of two emissions (644nm/584nm) and

pKa, Rmin and R ax from the in situ titration in the same regions ofthe cells in the fiirther

23

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data analysis with MS Origin® statistical software and Sigma Plot for Windows,

5.0 (Jandel Scientific, San Rafael, CA) (detail in data analysis).

version

Immunocytochemistry

Monoclonal antibodies to several subunits of V-H^ATPase are available from

commercial sources (e.g., 60 kDa, 69 kDa, and 100 kDa subunits; Molecular Probes,

Eugene, OR). Immunocytochemistry was performed on permeabilized cells as described

previously (Lynch et al. 1996). Briefly, cells were fixed with 4% paraformaldehyde for

15 min, washed with 25 mM glycine, and then permeabilized with 0.1% Triton X-100.

The cells were sequentially incubated with primary antibody which bound to the V-

H^ATPases, washed extensively, and then incubated with Texas Red-labeled secondary

(anti-mouse immunoglobulin G) antibody. Cells were subsequentiy incubated with

fluorescein-conjugated phalloidin (FITC-phalloidin) which bound to F-actin to delineate

the edges ofthe cells. Confocal microscopy images of five randomly selected areas per

coverslip from five independent experiments were analyzed. Simultaneously acquired

images of FITC-phalloidin (cytoskeleton) and Texas-Red (V-H^ATPase) fluorescence

were collected. Z-series scan enabled us to visualize the location and colocalization ofthe

proteins in a series of sections and each section was analyzed on a pixel by pixel basis

utilizing LaserSharp software (Bio-Rad) to assess colocalization of FITC and Texas-Red

at the plasma membrane.

26

Data Analysis

In situ calibration curves were generated as described previously (Martinez-

Zaguilan et al. 1991). Briefly, the cells attached to cover slips were perfrised with a high

K buffer (pH 5.5 to 8.0) containing 2 }iM valinomycin and 6.8 ^M nigericin. The high

K is used to approximate intracellular K and nigericin sets the H gradient equal to the

K"*" gradient, with valinomycin completing the collapse ofthe K" gradient without

significant effects on cell volume. The in situ titration for the single cell experiments,

including laser scanning confocal microscope (LSCM) and spectral imaging microscope

(SIM), were performed at the end of each experiment. After the ammonium chloride acid

loading procedure, the media in the chamber was removed and substituted with high K

buffer (pH 5.5) containing the same concentration of valinomycin and nigericin as above.

Then the media in the chamber were titrated with 0.5 M NaOH. The pH ofthe buffers

was determined using a Beckman pH meter with a glass electrode (Coming Inc.,

Horseheads, NY) calibrated at 37EC with commercially available standard solutions

(VWR Scientific, San Fransico, CA). Using the ratio (R=644/584) of SNARF-1 it was

possible to determine the R values at each pH studied during in situ calibrations. The

observed R-values were fit into the following equation:

p H = p K a + l 0 g [(Robs-Rmin)/(Rmax-Robs)] (1)

Where Robs was the ratio observed at any given pH, Rmin was die ratio observed when the

dye was ftilly protonated, and R ax represents the ratio of fluorescence obtained when the

27

H H I

dye was ftilly unprotonated. The equation was solved using nonlinear least squares

analysis with SigmaPlot to obtain the values of pKa, R m, and R ax for SNARF-1 in these

cells. These in situ calibration parameters were used to generate the pHi„ values for each

individual experiment by using equation (1) with SigmaPlot.

The initial rate of recovery from an ammonium chloride induced acid load was

measured as dpH/dt, as described previously (Roos and Boron 1981). Briefly, to

determine dpH/dt we looked at recovery of pH in the first five minutes following acid

loading. The individual data points were subtracted from the zenith pH at five minutes

and plotted against time. These points where then used to construct a linear regression

curve relating time and delta pH. The dpH/dt was expressed as the slope ofthe linear

regression as described previously (Martinez-Zaguildn et al. 1993). The apparent

buffering capacity (3i) is given by (Roos and Boron 1981):

A(apparcnt) = A[NH3]i/ApH (2)

where )[NH3]i is assumed to be equal to:

A [NH3]i = [NH3]o x{10 P' -P"'"V[l+10 P' -P"' ' ]} (3)

and )pH is the change in pHjn from the plateau after the NH4CI load and the nadir pHin

after NH4CI removal. To quantify the pHjn recovery, we expressed the recoveries as

proton fluxes (JH^) which is given by:

28

JH - pi (apparent) X dpH/dt. M)

Invasion and Migration Assay

Breast cells were grown to confluence in T-25 flasks in DMEM. At confluence,

the cells were loaded with 5 iM Calcein-AM for 30 minutes. Cells were then trypsinized,

washed, and counted. To evaluate the migration ability ofthe breast cancer cell lines,

cells were seeded on HTS FluoroBlok™ (Becton Dickinson, Bridgeport, NJ) at densities

of 1 X 10 . To evaluate the degree of cell invasion through various extracellular matrix

proteins (ECMs) in vitro, HTS FluoroBlok M inserts were briefly soaked in Matrigel

(Collaborative Biomedical Products, Bedford, MA). Inserts seeded with breast cancer

cells were incubated at 37EC in 5% CO2 for 48 hours in the presence or absence of 50

nM bafilomycin A|. HTS FluoroBlok' ^ inserts contain a 10 ^m proprietary polyethylene

terephthalate (PET) membrane impregnated with light absorbing dyes that will absorb

visible light from 490-700 nm. The inserts were subsequentiy visualized and images of

the bottom and top ofthe insert obtained with a 20X objective and a Bio-Rad 1024 MRC

confocal microscope (Bio-Rad, Hercules, CA). Cell counts from bottom images

compared to counts from top images were used to assess invasion. Calcein was excited

with the 488 nm line of a 15 mW argon laser and emission was collected utilizing the

VHS/open series filter blocks which contain an OG515 emission filter. Five images per

insert were obtained of experiments done in triplicate. The images were subsequently

29

analyzed and cells counted with the assistance of Scion Image (Scion Corporation,

Frederick, MD).

ATPase Assay

ATPase assays were done as described previously (Pressley et al. 1986). In brief,

breast cancer cells were grown till confluent in 100 mm petri dishes in L-15 media, and

then harvested, washed 3 times with homogenization buffer (10 mM tris-HCl, pH 7.4,

150 mM NaCl and 1 mM EGTA) and homogenized in a Dounce homogenizer (Sears

Roebuck and Co., Chicago, IL). The homogenates were centriftiged in 5000 xg, 4°C for 5

minutes with a Beckman J2-MC centriftige (Beckman Instruments Inc, Palo Alto, CA).

The supematants were remove to sealed 27 ml vials then centrifuged in 50.2Ti rotor

34100 rpm (50,000 xg), 4°C for 45 minutes with Beckman XL-96 Ultracentriftiges

(Beckman Instruments Inc, Palo Alto, CA). The pellets were washed and resuspended

with suspension buffer (50 mM trisHCl, pH 7.4, 250 mM sucrose, 1 mM EGTA).

Membrane fraction was obtained and protein concentration was determined by Lowry

assay (Lowry 1951) and then stored at -70°C. Protein (membranes fraction) was diluted

to 1 mg/ml, preincubated with or without (control) 50 nM of bafilomycin Ai for 20

minutes at room temperature. Detergent DOC (7%) was added into the solution (1:10)

and consequently incubated at 37°C for 30 minutes. The reaction was started by addition

20 \jLg of cell membranes to 200 pi solution containing 25 mM tris-HCl, pH 7.0, 0.2 mM

EGTA, 5 mM MgCh, 0.03 mM NaN3, 3.5 mM ATP (Sigma) and I-IOXIO"^ cpm of [y-

^"P] ATP (NEN Life Science Products), with or without 5 pM ouabain. After 60 minutes

30

in 37°C water bath, the reaction was stopped by addition of 1:1 percloric acid (20%

W/V):ammonium molybdate (8%) in ice. The samples were vortexed with iso-butanol,

cooled in ice, centriftiged and 500 pi supernatant was used to quantify the P released

during the incubation with a Beckman LS3801 Liquid Scintillation systems (Beckman

Instruments Inc, Fullerton, CA).

Statistical Analysis

Data are expressed as mean ± SE. Data were analyzed by paired /-tests where

indicated. Statistical significance was assigned at p values of < 0.05.

31

CHAPTER III

RESULTS

V-H""ATPases Colocalize with Actin Mainly in the Leading

Edge ofthe Cancer Cells

Previous studies have shown that V-H^ATPases are ftinctionally expressed in

plasma membrane of some invasive cells (Martinez-Zaguilan et al. 1993). However, the

location and the distribution of this enzyme in breast cancer cells are not clear. Using

immunocytochemistry we have shown the location ofthe V-H^ATPases. Breast cancer

cells were stained with monoclonal antibody against the 69 kDa subunit of V-H^ATPase,

then the second antibody, goat anti-mouse, conjugated with Texas-Red. Cells were also

co-stained with FITC-phalloidin to mark the cytoskeleton. The double photoamplifier

tube confocal microscope collected both the emission of Texas-Red and phalloidin.

Images were merged and colocalizations were analyzed as mentioned in Chapter II. Data

show that V-H^ATPases are expressed not only intracellulariy, but also on the surface of

the cells (Figure 5).

Colocalization of both V-H^ATPase and F-actin showed these two proteins

mainly colocalize on the cell surface, especially in the leading edge or lamellipodia ofthe

cells. Z-series scan ofthe cancer cells supported that tiie two proteins mainly colocalize

on the surface ofthe cells (data not shown). The position ofthe enzyme suggests that V-

H^ATPase may play a role in addition to pH regulation. Colocalizing with F-actin may

suggest the enzyme may involve in cell movement or maintaining cell shape.

32

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33

Bafilomycin A| Inhibits the Invasion ofthe Breast Cancer Cells

Invasion and migration are two prominent characteristics of tumor malignancy.

Many mechanisms maybe involved in these procedures. To evaluate the role of V-

H^ATPase in the migration and invasion activity ofthe breast cancer cells, invasion and

migration assays were performed. Cells were plated on top ofthe Fluoroblok inserts with

or without 50 nM bafilomycin Ai. Cell numbers were counted from both the top and the

bottom of the inserts.

The results of invasion and migration assays are shown in Table 2. MCF-7 cells

had the least migration and invasion number in all four cell lines tested, which is

statistically significant. No significant differences were found among MDA-MB-468,

MDA-MB-231 and MDA-MB-468 cells in migration and invasion.

Figure 6 shows migration (b) and invasion (a, c) in different breast cancer cell

lines with or without 50 nM bafilomycin Ai, a specific V-H""ATPase inhibitor. Invasion

cell numbers are reduced in all the cell lines and the differences are significant. IN

contrast, no significant difference was found in the migration assays with or without

bafilomycin Aiin any ofthe cell lines tested.

Counts ofthe cell numbers on the top ofthe inserts showed no significant

difference among all the cell lines. Nor was a significant difference found between with

and without bafilomycin A, in each cell line (data not shown). The results suggest that V-

H ATPase may involve in the invasion ofthe cancer cells.

34

Table 2. Results of invasion and migration assay (n=5)

Cell lines

MDA-MB-468

MDA-MB-231

MDA-MB-435S

MCF-7 Stars (*) show s

Invasion Cell number/insert

(mean ± SD) 10346 ±4513

24380 ± 17460

1319 ±10483

360 ± 153

Invasion + Baf Cell number/insert

(mean ± SD) 1066 ±403 •

746 ± 232 •

80.± 26 •

26 ± 26 • ignificant difference between with Baf (bafi

Migration Cell number/insert

(mean ± SD) 24773 ± 7309

38353 ± 19327

54133 ± 13583 733 ± 140

Migration + Baf Cell number/insert

(mean ± SD) 21946 ±6662

54506 ± 3982 10000 ±4240

1200 ±801

omycin A|) and without Baf (p<0.05)

Proton Fluxes Are Larger in Highly Invasive Breast Cancer Cells

So far, we know that V-H"" ATPases are expressed on the cell surfaces ofthe

breast cancer cells and maybe involved in the invasion procedures ofthe breast cancer. Is

the V-H""ATPase expression and their ftinction equal in different breast cancer cell lines?

To answer this question, acid loading experiments were carried out in cell groups ofthe

breast cancer cell lines. Cells were loaded with SNARF-1. Emissions of SNARF-1 were

measured with a spectrofluorometer. Cells were acidified by adding NH4CI to the media

and then the NH3 washed out with Na^ free and Ca ^ free CSB. Since Na^ free and Ca ^

free CSB prevented the action of all the Na^ dependent H^ transporters and exchangers,

Cells had to depend on the proton pump to remove the extra H"". Proton fluxes were

measured from the recovery phase ofthe NH4CI acid loading experiment as mentioned in

Chapter II. Results showed that MDA-MB-231 cells had larger proton fluxes than MCF-7

(Figure 7a, b), indicating that more proton pump activities existed in MDA-MB-231

cells.

35

MCF-7 Cells Mb231 CeUs MB468 Cells

Top of the inserts

Bottom of the inserts

No Baf

Bottom of the inserts

with Baf

g 10x103

^ 1X103 ^

> 100x10° •

2 10x10° { MB468 MB231 MB435S MCF-7 MB468 MB231 MB435S MCF-7

Figure 6. Bafilomycin A,(Baf) inhibits invasion ofthe breast cancer cells Cells were loaded with Calcein and plated in HTS Fluoro-Block inserts for 48 hours in the presence or abscence of 50nM bafilomycin A,. The uiserts were visualized under LSCM. Stars show significant difference between no Baf and with Baf (n =5, p<0.05).

36

NH.CI Na"- Free

8.0 .

MB231

MB435S MCF-7 ^

5 min

MCF-7

MB435S

IVB231

0.

B

1-H

-

0 0.3 0.6 0.9

J^ (nrMmin)

Figure 7. Highly Invasive Cells Exhibit Larger Proton Fluxes (JH+) Than Lewdly Invasive Cells. A. Acid loading experiments in cell populations. Cells v^ere loaded with SNARF-1. Tthe ratios of both emission 644/584 were measured. Intracellular pHs were calculated. B. Proton fluxes were quantified as mentioned in chapter II. Highly invasive breast cancer cells

have larger proton flux than lowly invasive(n=ll , differences are significant between groups, p<0.05). C. Bafilomycin Al inhibits proton flux in MB231 cells data are representative of 7 experiments..

37

In order to further confirm that proton fluxes were produced by V-H""ATPase,

bafilomycin Ai was introduced into the Na free and Ca free CSB. Data showed that

bafilomycin Ai reduced the proton fluxes in MDA-MB-231 cells (Figure 7c).

Combining these results with the data from the invasion and migration assays (MDA-

MB-23 1 cell is more invasive than MCF-7 cells), we conclude that highly invasive breast

cancer cells have more proton pump activities. These results also supported indirectly

that V-H^ ATPase maybe involved in the invasion ofthe cancer cells.

Intracellular pH Gradients Are Larger in Highlv Invasive

Breast Cancer Cells

It is well known that cancer cells live in an acidic environment and the pHin is

alkaline. We also know from the present study that V-H"^ATPases are expressed on the

plasma membrane ofthe breast cancer cells. The immunocytochemistry data indicated

that the distribution of this enzyme is not uniform. Based on this, we hypothesize that the

pHin of breast cancer cells is not evenly distributed either. To unveil this secret, line

scanning confocal microscopy (LSCM) was employed to measure the pH.n- In the present

study, we drew a line from the leading edge to the lagging edge ofthe cells. Emissions of

SNARF-1 were collected from four points on the line (Figure 3) every 2 milli-seconds.

Intracellular pH values were calculated as mentioned in data analysis. Chapter II. Data

showed that pHjn is not equally distributed in the breast cancer cells; i.e., pHin gradients

did exist. The pHin at the leading edge ofthe cell was more alkaline than that at the

lagging edge. Intracellular pH gradients were larger in the highly invasive cancer cells

38

Hd

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39

(Figure 8a), such as MD-MB-231 cells, than in the lowly invasive cancer cells, such as

MCF-7 (Figure 8b). Proton fluxes were larger in the leading edge than in the lagging

edge. This implied that more proton pump activities occurred in the leading edge ofthe

cancer cells and it was more prominent in the highly invasive cell line.

Proton Pump Activities Are Elevated in the Leading Edge ofthe Breast Cancer Cells

Spectral imaging microscope (SIM) can also measure the pHin ofthe single cell.

In this experiment, breast cancer cells were loaded with SNARF-1, single cell was chosen

under 60x oil objective the cell was orientated from the leading edge to the lagging edge.

First order images were obtained with a 610 nm emission filter. The entrance slit ofthe

spectrograph (Figure 2) was narrow down from 2000 pm to 200pm and the emissions

were collected by reading sixteen spectra from the slit. The pH measurements of 16

intracellular areas in the slit were obtained by the data analysis afterwards. The pH

gradients were consistent with our observation from the LSCM. The leading edge ofthe

cancer cell had a more alkaline pH than the lagging edge. In the recovery stage ofthe

NH4CI acid loading experiment, proton flux was larger in the leading edge than in the

lagging edge (Figure 9). This fiirther confirmed the results ofthe LSCM.

Bafilomycin Sensitive ATPase Activities Were Discovered in the Plasma Membrane Fractions ofthe Breast Cancer Cells

V-H^ATPases have been found on the cell membrane immunocytochemically and

physiologically in all the experiments in the present study. To more directly address this

40

issue, we then performed biochemical studies. Breast cancer cells were cultured to

confluency and harvested. After homogenization, the membrane fraction ofthe cells was

used for the ATPase assay. The ATPase activities measured from the membrane fraction

ofthe breast cancer cells without drug treatment served as the control (100%). In the

experimental groups, membrane fractions were preincubated with 50 nM bafilomycin Ai.

or with both 50 nM bafilomycin Ai and 5 pM ouabain. Results show that bafilomycin Ai

significanfly decreased the ATPase activity MDA-MB-468 and MDA-MB-231 cells as

compared with the control groups. Bafilomycin Ai sensitive ATPase activities composed

25 - 45% ofthe total ATPase activities on the cell membranes ofthe breast cancer cells.

Of membrane fractions preincubated with both bafilomycin Ai and ouabain (5 pM), the

ATPase activities tended to be even lower (Figure 10), which suggested that the

bafilomycin sensitive ATPase activity was something other than Na^, K"'-ATPase.

41

7.5

7.0

a 6.5-

6.0-

0 5 10 15 20 25 30 35 Time (min)

Figure 9. Spectral imaging microscope show intracellular pH gradients exist from leading edge to lagging edge ofthe MDA-MB231 breast cancer cells. The leading edge ofthe cell have a more alkaline pH.

42

CO CN

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cn

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43

^^^m^^

CHAPTER IV

DISCUSSION AND CONCLUSION

V-H ATPase and Tumor Invasive Behavior

Tumor invasion and metastasis are two hallmarks ofthe neoplasm malignancy.

They are the major causes ofthe morbidity ofthe cancer patients. Liotta et al. (1986)

delineated events related to the process of invasion ofthe extracellular matrix, including

the basement membrane, by tumor cells. These events include: (a) attachment of tumor

cells to laminin or fibronectin via cell surface receptors for these molecules; (b) local

degradation ofthe matrix by tumor cell-associated proteases; and (c) tumor cell

locomotion through the matrix, assisted by proteolysis. The invasive capacity ofthe

tumor cell can be examined in vitro by amnion invasion assay (Yagel et al. 1989) and

Matrigel assay (Hendrix et al. 1987). Many factors maybe involved in this process, such

as Ca2 (Marks et al. 1990, Milne et al. 1991, Savarese et al. 1992, Korczak et al. 1989),

chemoattractants (Miller et al. 1990, Mareel et al. 1990), and several proteases, e.g.

collagenase (Yagel et al. 1989, Stetler-Stevenson 1990), cathepsins (Lah et al. 1992,

Rozhin et al. 1994), metalloproteinases (Matrisian et al. 1992) and serine protease (Fidler

1991, Vassalli et al. 1991), etc. No direct evidence for V-H" ATPase involvement in

tumor invasion has been documented yet. However, the relation between proton

concentration (pH) and invasion has drawn extensive attention. Firstly, all the proteases

mentioned above are pH sensitive. Cathepsins are lysosomal enzymes, which prefer an

acidic pH (Morisset et al. 1986). Acidic pH induces the redistribution and release of

44

cathepsin B from a series of metastatic human cell lines (Rozhin et al. 1994).

Mathematical models have been used to investigate whether altered proteolytic activity at

low pH is responsible for the stimulation of a more metastatic phenotype. Webb et al.

(1999) examined the effect of culture pH on the secrefion and activity of two different

classes of proteinases: the metalloproteinases (MMPs) and the cysteine proteinases (such

as cathepsin B). The mathematical modeling suggested that changes in MMP activity at

low pH did not significantly affect invasive behavior. However, the model predicted that

the levels of active cathepsin B were significantly altered by acidic pH. This result

suggested a critical role for the cysteine proteinases in tumor progression (Webb et al.

1999). Acidic pH directly associated with invasive behavior of tumors was noticed

recently by Martinez-Zaguilan et al. (1996). In this research, the in vitro invasive

potential of two human melanoma cell lines, the highly invasive C8161 and poorly

invasive A375, were examined. Culturing of either cell line at acidic pH (6.8) caused

dramatic increases in both migration and invasion, as measured with the Membrane

Invasion Culture System (MICS). These data indicated that culturing of cells at mildly

7.+

acidic pH induced them to become more invasive. Intracellular calcium ([Ca ];„) was

also examined in this experiment but it was not consistent with invasive potenfial

(Martinez-Zaguilan et al. 1996). Tumor cells have a lower extracellular pH (pHcx) than

normal cells; this is an intrinsic feature ofthe tumor phenotype, caused by alterafions

either in acid exports from the tumor cells or in clearance of extracellular acid. Low pHex

benefits tumor cells because it promotes invasiveness, whereas a high pHin gives them a

competitive advantage over normal cells for growth. Molecular genetic approaches have

45

revealed hypoxia-induced coordinated upregulation of glycolysis, a potentially important

mechanism for establishing the metabolic phenotype of tumors (Stubbs et al. 2000). A

combined result of mathematical analyses, experimental data, and clinical observations

also supported the hypothesis that tumor-induced alteration of microenvironmental pH

may provide a simple but complete mechanism for cancer invasion (Gatenby et al. 1996).

Controversies also exist in this field. Cuvier et al. (1997) examined whether the

invasive capacity ofthe cells could be influenced by hypoxia, glucose starvation and

acidosis with a Matrigel, a basement membrane-like preparation, in a two-chamber

invasion assay to address this issue. Both KHT-LPl, a murine sarcoma cell line, and

SCC-VII, a murine squamous carcinoma cell line, showed an increased ability to invade

through Matrigel after hypoxia and glucose starvation, but there was no consistent change

in invasive capacity following acidosis exposure. They also found that cathepsin (L+B)

content varied according to the cell line and the treatment received (hypoxia, glucose

starvation). There was an increase of cathepsin content for KHT-LPl cells exposed to

hypoxia which correlated well with the increase ofthe invasion ability through Matrigel.

No increase of cathepsin content was observed for hypoxia-treated SCC-VII or for KHT-

LPl and SCC-VII cells treated with glucose starvation. These results suggested that

transient hypoxia and glucose starvation, but not acidosis, can increase the invasive

ability of tumor cell lines.

Evidence of V-H^ATPase involved in microvascular invasion was found recently

in our lab. Microvascular endothelial cells thrive in an acidic environment, suggesting

that they must exhibit a dynamic pH^ regulatory mechanism to cope with acidosis. The

46

results showed that these cells: (a) exhibited pmV-H""ATPase activity; (b) expressed

pmV-H"*"ATPase activity at the leading edge during migration; (c) exhibited a more

alkaline pHin at the leading edge of migrating cells; and (d) migrafion and invasion of

microvascular endothelial cells were inhibited by bafilomycin Ai. Altogether, these data

suggested that pmV-H" ATPases were essential for invasion and migration, the two

essential steps in angiogenesis (Rojas et al. 2001). V-H" ATPases proved important for

acidification-dependent degradation of tissue matrices, through which some cell types

move, and for pH regulation across some epithelial cell layers. Placentation involved

intricate signaling, cell proliferation, and controlled invasion. Redistribution V-

H^ATPase was observed and found to be stage-dependent during the bovine

implantation. Epithelial expression of all three subunits of V-H"* ATPase was observed,

and in nonpregnant animals this expression was apical. As pregnancy proceeded,

expression of all subunits became pericellular in luminal but not glandular epithelium.

The trophoblast expressed all three subunits during initial contact with the epithelium. In

the stroma, ductin (the 16kD subunit of V-H^ATPase) expression was reduced after

implantation. This suggested that ductin plays a role in the shifting communication

between stromal and epithelial cells induced by embryo attachment (Skinner et al. 1999).

In Kubota et al.'s study (2000), they established transfectants over-expressing the V-

H^ATPase 16 kDa subunit at the mRNA level, and found that these transfectants showed

an enhanced invasiveness through Matrigel with concomitant increases in secretion of

matrix metalloproteinase-2. Moreover, antisense oligonucleofides ofthe V-H" ATPase 16

kDa subunit suppressed invasive human A549 cell invasion with concomitant decreases

47

in secretion of matrix metalloproteinase-2. The results suggested that the V-H*ATPase 16

kDa subunit is directly involved in cell invasion and that matrix metalloproteinase-2 is

responsible for promoting the invasion by the V-H^ATPase 16 kDa subunit.

Overexpression of a single subunit of V-H^ATPase does not reflect the real

activity ofthe enzyme. In the present study, we studied the intact cancer cells in order to

determine the physiological role of V-H^ATPase. The immunocytochemistry data

showed that V-H^ATPases were expressed on the cell's surface predominanfly in the

leading edge. The J ^ was found elevated more in highly invasive cells than in lowly

invasive breast cancer cells by the spectrofluorometer experiments. Both spectral imaging

and confocal line scan data showed that breast cancer cells exhibited a more alkaline pH

in the leading edge than in the lagging edge. Intracellular pH gradients were larger in the

highly than in the lowly invasive cells. These findings suggest that the highly invasive

breast cancer cells have more V-H^ATPase activifies than the lowly invasive ones.

Bafilomycin A, inhibited the invasion of metastatic cells, implying that V-H*ATPase

maybe involved in the invasion procedure ofthe breast cancer cells.

In the past, it was believed that proton-motive forces, generated from ATP by V-

H"* ATPases, were less predominant than sodium-motive forces, generated by P-ATPases

as energizers of animal membranes. About one decade ago, Anraku (1989), Nelson

(1992), and others showed that proton-motive forces from V-H^ATPases energized

endomembranes of all eukaryotic cells; in most cases, chloride ions accompanied the

protons and the output compartment was acidified. Unexpectedly, numerous examples of

animal plasma membrane energization by proton-motive forces are now appearing. In

48

many insect epithelia, V-H^ATPases generate transmembrane voltages which secondarily

drive sensory signaling, fluid secretion and even alkalization, rather than acidification.

Plasma membranes of phagocytes (Swallow et al. 1990) and osteoclasts (Blair et al.

1989), as well as polarized membranes of epithelia in vertebrate kidney (Gluck 1992),

bladder and epididymis (Brown et al. 1992) and also cancer cells (Martinez-Zaguilan et

al. 1993) are now known to be energized by proton-motive forces. The list of proton-

energized animal plasma membranes continues to grow (Harvey et al. 1997). Does

proton-motive force in plasma membrane ofthe tumor cells help the cell move or

metastasize easier? To answer this question more research needs to be done.

Vacuolar H^ATPase and Tumor Metastasis

So far, no sufficient evidence has been documented that suggests V- H ATPases

are directly related to metastasis. Our work suggests that V-H^ATPases are involved in

tumor metastasis. The previous experiments of this lab showed that human melanoma

cells with different metastatic potential have distinct pHjn and [Ca ]in regulation

mechanisms (Martinez-Zaguilan et al. 1998). V-H^ATPases are functionally expressed at

the plasma membranes of highly metastatic, but not in pooriy metastatic human

melanoma cells (Martinez-Zaguilan et al. 1993).

The recent study of Thomsen et al. (1999) demonstrated that the inhibition ofthe

V-H^ATPase in NIH 3T3 cells with bafilomycin Ai or by transfection of cells with the

HPV-16 E5 oncogene, which binds to the 16kD subunit of V-H""ATPase and interferes

with its ftinctioning (Hwang et al. 1995, Yamshchikov et al. 1995), led to a changed

49

morphology and a reduced motility. Bafilomycin A, potentiated the effect ofthe E5

protein on cell mofility and this cooperative effect indicated that the E5 protein and

bafilomycin A, either targeted the V-H"ATPase differently or that the E5 protein had

additional targets in transfected cells. The data suggested that proper ftinction ofthe V-

H ATPase is needed for normal cell locomotion.

In osteoclasts, the cells that degrade bone, V-H^ATPases, were recruited from

intracellular membrane compartments to the ruffled membrane, a specialized domain of

the plasma membrane, where they were maintained at high densities, serving to acidify

the resorption bay at the osteoclast attachment site on bone (Blair et al. 1989). V-

H^ATPases in osteoclasts cultured in vitro were found to form a detergent-insoluble

complex with actin and myosin II through direct binding of V-H^ATPase to actin

filaments. Plating bone marrow cells onto dentine slices produced a profound change in

the association ofthe V-H^ATPase with actin, assayed by coimmunoprecipitation and

immunocytochemical colocalization of actin filaments and V-H^ATPase in osteoclasts.

Mouse marrow and bovine kidney V-H^ATPase bound rabbit muscle F-actin direcUy

with a maximum stoichiometry of 1 mol of V-H"* ATPase per 8 mol of F-actin and an

apparent affinity of 0.05 pM. Electron microscopy of negatively stained samples

confirmed the binding interaction (Powell et al. 2000). These findings linked transport of

V-H^ATPase to reorganization ofthe actin cytoskeleton during osteoclast activation.

Actin and myosin are two important components of cell movement (Albert et al. 1994).

Montcourrier et al.(1994) examined the large acidic vesicles of human metastatic

breast cancer cells in culture by transmission electron microscopy, which measured pH of

50

the vesicles by video-enhanced epifluorescence using FITC-dextran. They found that the

presence ofthe large vesicles in metastatic MDA-MB-231 cells correlated with an

increased ability of cells to migrate through Matrigel and a high cathepsin D

concentration. These cells were able to phagocytize 1.24-micron latex beads and

fluorescent Matrigel and incorporate this extracellular material into large acidic vesicles.

Large acidic vesicles were actively acidified with an H^-ATPase vacuolar pump

specifically inhibited by bafilomycin A]. This indicated that large acidic vesicles were

associated with both phagocytosis and invasion. Montcourrier et al concluded that the

phagocytotic activity of breast cancer cells, associated with high cathepsin D expression

and high acidification potential, characterized cancer cells that migrated through

Matrigel.

In conclusion, the present study showed that more V-H^ATPases are expressed at

the surface ofthe highly invasive breast cancer cells than in the lowly invasive cells.

Invasion of metastatic cells was inhibited by bafilomycin Ai a V-H+ATPase inhibitor.

The J ^^ was more elevated in highly invasive cells than in lowly invasive breast cancer

cells. Breast cancer cells exhibited a more alkaline pH in the leading edge than in the

lagging edge. Intracellular pH gradients were larger in the highly than in the lowly

invasive cells. A complete understanding ofthe mechanism by which V-H"" ATPase

works in invasion and metastasis require ftirther study. However, energization ofthe

movement of molecules across the cell membrane and changing the pHi„ consequenfly

activates the proteins related to cell motility maybe one ofthe possible mechanism.

51

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