Characterization of Expression in Multiple Myeloma and ... · Vol. 3, 2173-21 79. November 1997...

Vol. 3, 2173-21 79. November 1997 Clinical Cancer Research 2173

Characterization of �16��(4A Expression in Multiple Myeloma and

Plasma Cell Leukemia’

Mitsuyoshi Urashima, Gerrard Teoh,Atsushi Ogata, Dharminder Chauhan,

Steven P. Treon, Yoshihisa Sugimoto,

Chiharu Kaihara, Masaharu Matsuzaki,

Yasutaka Hoshi, James A. DeCaprio, andKenneth C. Anderson2

Divisions of Hematologic Malignancies IM. U.. G. T., A. 0.. D. C..

S. P. T.. K. C. A.l and Neoplastic Disease Mechanisms Ii. A. Dl.Dana-Farber Cancer Institute and the Department of Medicine.

Harvard Medical School, Boston, Massachusetts 02215; Research and

Development Center for Molecular Biology and Cytogenesis lY. S.land Cellular Therapies Unit IC. K.. M. M.], SRL. Inc., Tokyo. Japan;

and Department of Pediatrics. Jikei University School of Medicine.

Tokyo. Japan [Y. H.l

ABSTRACTLoss of p161NK4A (p16) expression is frequently associ-

ated with the development of epithelial and lymphoid ma-

lignancies. However, the frequency and significance of p16abnormalities in multiple myeloma (MM) and the more

aggressive phase of plasma cell leukemia (PCL) have notbeen well defined. Accordingly, the goal of this study was to

define the expression and function of p16 in fresh samples of

MM and PCL. We found that p16 protein was highly ex-pressed in primary MM cells, although it was undetectable

in fresh samples of PCL. Additionally, p16 protein was also

absent in four of four MM-derived cell lines. To determine

the mechanism for p16 underexpression in PCL and MM-

derived cell lines, we performed PCR analysis to evaluate

both gene deletion and the presence of methylation. Inter-estingly, the p16 gene was present and methylated in all

patient PCL cells and MM cell lines, whereas it was un-

methylated in patient MM cells and normal B cells. Further-

more, treatment with the demethylating agent 5-deoxyaza-cytidine or p16 retrofection restored p16 protein expression

and induced G1 growth arrest in patient PCL cells and MM

cell lines. These results suggest that inactivation of the p16

gene by methylation may be associated with decreased

growth control and the development of PCL in a subset ofpatients with MM.

Received 3/4/97: revised 7/18/97: accepted 8/8/97.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

� Supported by NIH Grant CA 50947.

2To whom requests for reprints should be addressed, at Dana-FarberCancer Institute, 44 Binney Street. Boston. MA 02215. Phone:

(617) 632-2144; Fax: (617) 632-2569; E-mail: [email protected].

INTRODUCTIONAbnormalities in cell cycle control are common in cancers

of solid organs as well as hematological malignancies. In par-

ticular, phosphorylation of pRB3 and related G1-to-S phase

transition is triggered by activation of cyclin D-CDK4/CDK#{212} or

cyclin E-CDK2 complexes. Conversely. CDIs such as

(pl6), p15 1NK4B (plS), or pl8�”’4� (pl8) compete with cyclin

D for binding to CDK4/CDK6 and. therefore. inhibit CDK4/

CDK6 complex kinase activity, resulting in dephosphorylation

of pRB and related growth arrest ( I ). Lack of functional

CDIs due to gene deletion, mutation, or methylation may result

in dysregulation of the cell cycle in cancer (2). For example. p16

gene-deficient mice develop spontaneous tumors including

lymphoid malignancies at an early age (3). In humans, p16 gene

is altered in both freshly isolated tumors and derived cell lines

(4-8). Within the hematological malignancies, homozygous

deletion ofpl6 gene occurs in 70-80% cases ofT-cell ALL and

in 15-20% of B-cell ALL (9-16). In this setting. p16 gene

deletion is associated with poor prognostic features. including

bulky disease, higher WBC counts, and higher relapse rates.

Within the blast crisis of chronic myelogenous leukemia, ho-

mozygous deletion of p16 gene has been reported in 40% of

lymphoid, but not in myeloid, subtypes (17). In contrast. dde-

tion of p!6 gene is rarely observed in acute myelogenous

leukemia, the chronic phase of chronic myelogenous leukemia,

myelodysplastic syndrome. chronic lymphocytic leukemia, or

MM (11-16, 18, 19).

Another mechanism for loss of CDI protein function is de

tiovo methylation of S’CpG islands in their gene promoters and

related transcriptional inactivation, as has been shown for p16

gene in non-small cell lung cancer, head and neck squamous cell

carcinoma, high-grade glioma, and colon cancer (20-22). DNA

methylation at cytosine located 5’ to guanosine in the CpG

dinucleotide can in some cases be related to increased levels of

DNA methyltransferase (23), and silencing of tumor suppressor

genes by methylation at CpG islands has now been reported in

several tumors: retinoblastoma gene in retinoblastoma, von Hip-

pd Lindau gene in renal carcinoma, and p53 gene in bladder

carcinoma (24, 25). Moreover. abnormal methylation of the

CpG island in certain genes correlates with structural changes in

chromatin and enhanced susceptibility to chemical carcinogens

(26, 27), and CpG sites are known hot spots for mutation (28.

29). These studies implicate aberrant gene methylation in car-

cinogenesis. The observations that CpG methylation is common

3The abbreviations used are: pRB. retinoblastoma protein: CDK. cy-

din-dependent kinase: CDI. CDK inhibitor: ALL. acute lymphoblastic

leukemia: MM. multiple myeloma; PCL. plasma cell leukemia: MC.

mononuclear cell; MoAb, monoclonal antibody: IP. immunoprecipita-

tion: WB. Western immunoblotting: 5-Aza-CdR. 5-deoxyazacytidine:

P1. propidium iodide.

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2174 p16 in Multiple Myeloma

in tumors but rarely observed in normal cellular counterparts

(30), coupled with decreased neoplasia observed in methyltrans-

ferase depleted mice (3 1), further support the role of gene

methylation in tumorigenesis.

Within the hematological malignancies. a recent study has

found distinct patterns of inactivation of CDIs p15 and p16, due

to homozygous deletion and/or hypermethylation (32). For cx-

ample. hypermethylation of p16. often without alterations of

p15, was found in non-Hodgkin’s lymphoma; in contrast, hy-

permethylation of p15 without alteration of p16 was an almost

universal finding in adult acute myelogenous leukemia. In T-cell

acute lymphoblastic leukemia, a high frequency ofpl5 and p16

gene deletions, coupled with hypermethylation of p15 gene but

not pitS gene, has been observed (33). Finally, a recent study of

MM has also shown hypermethylation of p16 and p15 genes to

be present in 75 and 67% cases, respectively (34).

In the present study, we examined p16 function in plasma

cell dyscrasias. p16 protein was highly expressed in MM cells

but was undetectable in patient PCL cells and MM-derived cell

lines. Although p16 gene was not deleted in patient PCL cells

and MM-derived cell lines, it was methylated in these cells; in

contrast, no p16 gene methylation was present in patient MM

cells. Restoration of p16 protein in malignant plasma cells either

by treatment with a demethylating agent or by p16 retrofection

induced growth arrest. These results demonstrate that p16

gene methylation contributes to decreased p16 protein and

growth control in plasma cell dyscrasias.

MATERIALS AND METHODSPatient MM Cells, Patient PCL Cells, and MM-derived

Cell Lines. MCs were isolated from bone marrow of eight

patients with MM and peripheral blood of three patients with

PCL by Ficoll-Hypaque density gradient centrifugation and

incubated with HB7 (anti-CD38) MoAb-biotin-streptavidin and

2H4 (anti-CD45RA) MoAb-FITC on ice. Tumor cells were

isolated using an Epics C cell sorter (Coulter Electronics,

Hialeah, FL; 96 ± 2% CD38 + CD45RA-), washed, and

resuspended in RPMI 1640 (Sigma Chemical Co. St. Louis,

MO) containing 10% fetal bovine serum, L-glutamine (Life

Technologies, Inc., Grand Island, NY), 100 units/ml penicillin,

and 100 p.g/ml streptomycin (Life Technologies, Inc.; Ref. 35).

The ARH-77, RPMI-8226, and U-266 human MM-derived cell

lines, as well as a pRB-deflcient SAOS-2 osteosarcoma cell line,

were obtained from American Type Culture Collection (Rock-

ville, MD). The OCI-MyS MM cell line was kindly provided by

Dr. H. A. Messner (Ontario Cancer Institute, Toronto, Ontario,

Canada; Ref. 36). The LPIO1 recombinant SV4O-adenovirus

transformed BMSC line was kindly provided by Dr. Shin

Aizawa (37). We have previously described the JKB ALL cell

line (38), which has a chromosomal translocation between 9p2l

(locus of pl6) and 14q32 (locus of heavy chain immunoglobulin).

B-Cell Preparation and Culture. Normal spleen was

obtained from operative specimens of patients not known to

have any systemic or malignant diseases. Single-cell suspen-

sions from spleen were prepared by extrusion through sterile

stainless steel mesh. Splenic MCs were isolated by Ficoll-

Hypaque density sedimentation, and adherent cells were re-

moved from MCs by double adherence to plastic Petri dishes for

I h at 37#{176}C.Further enrichment for B cells in spleen was done

by rosetting with sheep RBCs to deplete T cells. B cell-enriched

fractions (>90% CD2O+) were cultured in RPMI 1640 con-

taming 10% fetal bovine serum, L-glutamine, and penicillin/

streptomycin, as described previously (39).

Immunoprecipitation and Western Immunoblotting.

IP and WB were performed as reported previously (40, 41). For

IP, cells (1 X 10� cells/sample) were washed three times with

PBS and lysed for 30 mm at 4#{176}Cin buffer: I mrs�i Tris-HC1 (pH

7.6), 150 mM NaC1, 0.5% NP4O, S mr�i EDTA, 1 msi phenyl-

methylsulfonyl fluoride, 200 p.M Na3VO4, aprotinin, and I msi

NaF. Anti-p16 MoAb (PharMingen, San Diego, CA) or anti-

CDK4 polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz,

CA) were added for 18 h at 4#{176}Cto immunoprecipitate protein

complexes. Proteins were collected using protein G-Sepharose.

When anti-pl 6 MoAb was used, protein G-Sepharose was pre-

treated with rabbit anti-mouse immunoglobulin 0 (Cappel Or-

ganon, Durham, NC). Aliquots of each lysate were analyzed by

SDS-PAGE. Proteins were transferred onto polyvinylidene di-

fluoride transfer membrane (NEN Dupont, Boston, MA), and

nonspecific binding was blocked by incubation with 5% skim

milk. The membrane was probed with these same antibodies

followed by anti-mouse or anti-rabbit immunoglobulin antibod-

ies conjugated with horseradish peroxidase (Amersham Corp.,

Arlington Heights, IL). Complexes were detected using the

enhanced chemiluminescence system (Amersham).

PCR for p16 Gene. PCR was performed using an Om-

niGene (Marsh Biomedical, Rochester, NY) with 100 ng of

genomic DNA, 40 �M of sense and antisense primers, 200 p.M

each of deoxynucleotide triphosphate, 1 X amplification buffer,

I .5 mr�i MgC12, 0.5 p.1 (2.5 units) Taq polymerase (Roche,

Branchburg, NJ), and 5% DMSO in a reaction volume of 25 p.1.

Amplification consisted of 94#{176}Cfor 2 mm, followed by 30

cycles of 94#{176}Cfor 30 s, 55#{176}Cfor 1 mm, and 72#{176}Cfor I mm.

Primers for amplifying p16 exon 2 were 5’-GCT TCC iTT

CCG TCA TGC CG-3’ and 5’-GGA CTG ATG ATC TGA

GAA TTTG-3’. As a control, 3-globin sequences were ampli-

fled using the following oligonucleotides: sense primer, 5’-AAC

AGA CAC CAT GGT GCA CC-3’; and antisense primer,

5’-CTA AGG TGA AGG CTC ATG GC-3’. The resulting PCR

products were electrophoresed on an ethidium bromide stained

3.0% agarose gel. The size ofpl6 and �-globin products are 393

and 362 bp, respectively.

Methylation-specific PCR. The methylation status of

CpG islands of p16 gene was determined using methylation-

specific PCR, as reported by Herman et a!. (42). DNA (1 p.g) in

50 p.1 was denatured by NaOH (final concentration, 0.2 M),

modified with 10 mM hydroquinone (Sigma) and 520 p.1 of 3 M

sodium bisulfite (Sigma) at pH 5.0, and incubated at 50#{176}Cfor

16 h. Modified DNA was purified using Wizard DNA purifica-

tion resin (Promega Corp., Madison, WI), modified by NaOH

(final concentration, 0.3 M) at room temperature for S mm,

precipitated with ethanol, and resuspended in water. Bisulfite-

modified DNA was amplified with primers (300 ng each per

reaction) as follows: methylated p16 gene sense 5’-TTATTA-

GAGGGTGGGGCGGATCGC-3’ and antisense 5’-CCAC-

I � or unmethylated pitS gene

sense 5’-1TATTAGAGGGT000GTGGATfGT-3’ and anti-

sense 5’-CCACCTAAATCAACCTCCAACCA-3’ . PCR was



P16>1

�globin

Clinical Cancer Research 2175

F4� ‘-,

‘-‘V � � � � “V ‘� � �o ‘�‘ � � �‘�#{176} �‘

1!�#!��! � � � I

p16 . �

CDK4 �.. e� ��jy � = ,� � �l?TI� �

Fig. I Expression of p16 protein in patient MM cells, patient PCL cells. and MM-derived cell lines. Total lysates obtained from LPIOI5V40-transformed BMSCs: SAOS-2 RB-deficient osteosarcoma cells: normal splenic B cells: patient MM cells (MM-/-MM-8) and patient PCI. cells

(PCL-1-PCL-3): OCI-My5. U-266. ARH-77. and RPMI-8226 MM cells: and JKB-ALL cells were immunoprecipitated with anti-pIfi MoAb followedby WB with the same MoAb. IP and WB with anti-CDK4 polyclonal antibody served as a control.

ri�

�Lnr��

L.N� i_�t��Ce?

�

� _____�

Fig. 2 Presence of p16 DNA and p16 mRNA in patient PCL cells and

MM cell lines. PCR was performed using DNA from patient PCL cells

and MM-derived cell lines. as well as JKB ALL cells (negative control).with amplification using primers for p16 exon 2 (393 bp) and �3-globingene (362 bp).

performed with 100 ng of genomic DNA, 300 ng of sense and

antisense primers, 1 .25 m�i of each deoxynucleotide triphos-

phate, I x amplification buffer, I .5 m�i MgC12. and 0.5 p.1 (2.5

units) Taq polymerase (Roche) in a reaction volume of 50 p.1.

Amplification consisted of 95#{176}Cfor 30 s, followed by 35 cycles

at 95#{176}Cfor 30 s, 60#{176}C(unmethylated) or 65 #{176}C(methylated) for

30 s, followed by 72#{176}Cfor 30 s. The resulting PCR products

were electrophoresed on an ethidium bromide-stained 3% aga-

rose gels. The size of both methylated and unmethylated p16

products are 234 bp.

Culture of Patient MM and PCL Cells as well as MM

Cell Lines with 5-Aza-CdR. To determine the biological

significance of methylation, patient MM and PCL cells as well

as MM cell lines (5 X l05/ml) were cultured with or without the

demethylating agent 5-Aza-CdR (I p.M; Sigma) for 3 days.

Changes in p16 protein expression and cell cycle distribution

were determined by IP/WB and P1 staining, respectively.

Ectopic Expression of p16 Gene in MM Cell Lines.

Full-length p16 cDNA in pCDNA3 (Invitrogen, San Diego.

CA), provided by Dr. Geoffrey I. Shapiro (Dana-Farber Cancer

Institute, Boston MA), was cloned into the EcoRIJSa!I site of

pBabe puro, provided by Dr. Mark Ewen (Dana-Farber Cancer

Institute: Refs. 43 and 44). pBabe-puro and pBabe-pl#{244}-puro

were introduced into Bing packaging cells, obtained from Dr.

Shapiro (Dana-Farber Cancer Institute), using standard calcium

phosphate transfection techniques (45). Bing cells were cultured

for 24 h after transfection, and 60% of supernatant was cx-

changed with fresh media for an additional 24 h. Then Bing cells

were cultured with puromycin (2.0 p.g/ml: Sigma) for 2 weeks.

After selection with puromycin and washing. supernatants were

obtained from 48-h cultures of Bing packaging cells transfected

with pBabe-puro or pBabe-puro-pl6. filtered (0.45 p.m). and

diluted ( I :2) with fresh media. MM-derived cell lines were next

cultured in pBabe-puro or pBabe-puro-p 16 supernatant for 48 h.

followed by 24-h culture in fresh media: this 72-h process was

done a total of three times. Selection for transfected MM cell

lines was then performed by culture with puromycin (2 p.g/rnl).

Cell Cycle Analysis. The effect of ectopic p16 expres-

sion in MM cell lines and of treatment of MM cell lines with the

demethylating agent 5-Aza-CdR on cell cycle distribution was

examined. Cell cycle analysis was done using P1 staining and

fluorescence-activated cell sorting analysis. as in previous re-

ports (46). MM cell lines were collected and suspended in 0.5

ml of 3.4 mM sodium citrate. 10 m�i NaCI, 0.1% NP4O. and 50

mg/mI P1 to stain nuclear DNA. Cell cycle distribution for each

sample (I x l0� cells) was determined using the Coulter Epics

753 cell sorter and program M Cycle software (Coulter Elec-

tronics).

RESULTS

Expression of p16 Protein in Patient MM Cells, Patient

PCL Cells, and MM-derived Cell Lines. Expression of p16

protein in patient MM cells, patient PCL cells. and MM-derived

cell lines was analyzed by IP and WB and compared with p16

expression in LP1OI SV4O-transformed BMSCs. RB-deficient

SAOS-2 osteosarcoma cells, or normal splenic B cells (Fig. I).

p16 protein was expressed in eight patient MM cells but at much

lower levels than in LPIOI or SAOS-2 cells. Although small

amounts of pl6 protein were detectable in normal B cells. it was

undetectable in three patient PCL cells. p16 protein was also

undetectable in all MM cell lines (OCI-MyS, U-266, ARH-77,



A

B


ARH- OCI- RPMI-J!� BedI �ll M�a M� � PCL2 � 77 MyS 8226 U�UM UM UM U M U M UM UMUM UM UM U M UM

234bp. �

Fig. 3 Methylation status of plO gene in patient MM and PCL cells as well as MM cell lines. p16 gene methylation was assessed using

methylation-specific PCR. DNA from JKB ALL cells, normal B cells, patient MM cells (MMI-MM3), patient PCL cells (PC’L1-PCL3), and MM

derived cell lines (ARH-77, OCI-My5. RPMI-8226, and U-266) were treated with bisulfite and amplified with primers for unmethylated (U� andmethylated (M) p16 genes.

and RPMI-8226) and in JKB ALL cells (37), which have ho-

mozygous deletion ofpl6 gene and served as a negative control.

CDK4 immunoblot analysis of the same cell lysates served as a

control.

Presence of p16 DNA in Patient PCL Cells and MM

Cell Lines. Because p16 protein was not detected in patient

PCL cells and MM-derived cell lines, we next probed for p16

DNA using PCR (Fig. 2). p16 gene was homozygously deleted

in JKB ALL cells, which served as a negative control. p16 gene

was present in SAOS-2 osteosarcoma cells, which served as a

positive control, as well as in normal B cells. p16 gene was

present in PCL patient cells and all MM-derived cell lines

(ARH-77, OCI-My5, RPMI-8226, and U-266). �3-globin DNA

was detectable in all samples.

Methylation ofpl6 Gene in Patient MM and PCL Cells

as well as MM Cell Lines. Because pitS gene was present but

p 16 protein was undetectable in patient PCL cells and MM-

derived cell lines, we next assayed for p16 gene methylation.

Specifically, we analyzed genomic DNA obtained from patient

PCL cells and MM cell lines using methylation-specific PCR

(42). As can be seen in Fig. 3, pitS gene was not methylated in

normal B cells and patient MM cells. However, both methylated

and unmethylated p16 gene was evident in patient PCL cells and

all MM cell lines except for U266. Only methylated p16 gene

was present in U266 cells. Neither unmethylated nor methylated

��/6 gene was present in JKB ALL cells.

Effect of Demethylating Agent on p16 Protein Expres-

sion and Cell Cycle Distribution in Patient MM and PCL

Cells as well as MM-derived Cell Lines. To confirm the

biological significance of pitS gene methylation, patient MM

and PCL cells, MM-derived cell lines, and JKB ALL cells were

next cultured with the demethylating agent 5-Aza-CdR ( I p.M

for 3 days); changes in expression of p16 protein and in cell

cycle distribution were analyzed using IP followed by WB and

P1 staining, respectively. p16 protein was induced by treatment

with 5-Aza-CdR in patient PCL cells (Fig. 4A) as well as in MM

cell lines (OCI-My5, RPMI-8226, and ARH-77; Fig. 4B) but not

in JKB p16 gene-deleted ALL cells. p16 protein was expressed

in patient MM cells and was unchanged by culture with 5-Aza-

CdR. As can be seen in Table 1, the percentage of patient PCL

cells in G1 increased 1 1-14% after treatment with 5-Aza-CdR,

but that of patient MM cells was not altered. The percentage of

MM cell lines in G1 also increased 12-14% in the presence of

5-Aza-CdR; in contrast, treatment of JKB ALL cells, with

deletion ofpl#{243} gene. did not significantly alter the percentage of

cells in G1.

MM! PCL1 PCL2 PCL3

5-Aza-CdR - + - #{247} - + - +

� - � - � �p16 � �

CDK4� II;r;E �OCI- RPMI- ARHMy5 8226 -77 JKB

5-Aza-CdR - + - + - + - +

� - - � � � -

p160

CDK4� �ir’�rnFig. 4 Effects ofculture with 5-Aza-CdR on p16 protein expression in

patient MM and PCL cells as well as MM-derived cell lines. Either 5 X

l05/ml patient MM and patient PCL cells (A) or MM cell lines (OCI-MyS. RPMI-8226, and ARH-77) and JKB ALL cells (B) were culturedwith or without 5-Aza-CdR (1 fi.M) for 3 days. Cell lysates wereanalyzed by IP and WB using anti-p16 Ab. IP and WB of cell lysates

with anti-CDK4 antibody served as a control.

Effects of Ectopic Expression of p16 in MM-derivedCell Lines. To confirm the function ofpl6 protein in MM cell

lines, p!6 gene was ectopically expressed in these cells using

retrofection with pBabe p 16-puro vector, followed by puromy-

cm selection. p16 protein was detectable in OCI-MyS and

RPMI-8226 MM cells transfected with pBabe p16-puro but not

in these cells transfected with control pBabe puro (Fig. 5). As

can be seen in Table 2, >20% increments in the percentage of

cells in G1 were observed in p16-transfected MM cells in

comparison with MM cells transfected with control vector.

DISCUSSION

In the present study. we demonstrated that p16 protein was

highly expressed in all patient MM cells but was undetectable in

patient PCL cells and MM-derived cell lines. Homozygous p16




Table I Effect o f 5-Aza-CdR on cell cycle distribution’� of tumor cells

Percentage of cells in phase of cell cycle”

MM1 PCL1 PCL2 PCL3

Patient cell - + - + - + - +

G

SG,-M

86

5

9

89

5

6

76 89 73

12 5 11

12 6 16

84

7

9

70

17

13

81

4

15

OCI-MyS RPMI-8226 ARH-77 JKB

MMcellline - + - + - + - +

G1

S

G,-M

41

33

26

53

15

32

46 59 50

40 31 31

15 10 19

64

20

16

50

29

21

57

19

24

‘, Cell cycle distribution (%) was analyzed by P1 staining followed by fluorescence-activated cell sorting.‘, -, without 5-Aza-CdR: +, with 5-Aza-CdR.

p16 �

OCI-My5 RPMI-8226

Vec p16 Vec p!6� - -

=

CDK4 0..

Fig. 5 Effects of ectopic expression of p16 in MM-derived cell lines.

p16 was ectopically expressed in MM-derived cell lines (OCI-MyS andRPMI-8226) by retrofection of pBabe-p16-puro vector (p16) or pBabe-puro control vector (Vec). After selection of transfected cells in puro-

mycin (2 pg/mI). cell lysates were analyzed by IP and WB using

anti-p16 Ab. IP and WB ofcell lysates with anti-CDK4 antibody servedas a control.

gene deletion and transcriptional inactivation of p16 gene asso-

ciated with methylation on the 5’CpG island region are the two

most common mechanisms for loss of p1 6 function in a variety

of cancers (2-34), although point mutation in p16 gene is

commonly noted in germ-line DNA obtained from patients with

familial melanoma (8). In hematological malignancies such as

ALL, plo gene deletion is a common reason for loss of p16

function (9-17, 32,33). In our study, no homozygous deletion of

pitS gene was noted in either patient PCL cells or MM-derived

cell lines, consistent with previous reports that deletion of pItS

gene is infrequent in clinical samples obtained from patients

with MM (15, 16, 18, 19, 34). For example, homozygous

deletion of p15 and pitS genes in a single case of PCL and

homozygous deletion of both p15 and p18 genes were observed

in 1 of 19 MM samples examined in a recent study (19).

To assay for methylation ofpl6 gene in patient MM cells,

patient PCL cells, and MM cell lines, we used methylation-

specific PCR. This method is based upon the conversion of

cytosine, but not 5-methylcytosine, to uracil by bisulfite treat-

ment; it eliminates the frequent false-positive results due to

Table 2 Cell cycle distribution” of p16 gene-transfected cells

Percentage of cells in phase of cell cycle

OCI-My5 RPMI-8226

Vector p16 Trans.” Vector p16 Trans.

G 43.00 64.70 46.30 69.30

S 36.80 23.20 37.70 22.40

G,-M 20.20 12.10 16.00 8.30

(‘ Cell cycle distribution (%) was analyzed by P1 staining followedby fluorescence-activated cell sorting.

,,Trans., transfected.

partial digestion of methylation-sensitive enzymes inherent in

previous methods for detecting methylation (42). Using meth-

ylation-specific PCR, we found only unmethylated pitS gene in

normal B cells and patient MM cells. Both unmethylated and

methylated p16 gene were present in patient PCL cells and

MM-derived cell lines, except for U-266 in which only meth-

ylated p16 gene was present. Because the extent of transcrip-

tional repression has been correlated with CpG methylation

density (47, 48), partial methylation of p16 gene observed in

patient PCL cells and MM cell lines may inhibit, but not

completely block, p16 gene transcription. In contrast, pitS gene

methylation in U-266 cells is sufficient to block pitS gene

transcription. Our data, therefore, demonstrate that methylation

can contribute to loss of p16 protein in PCL cells. Further

supporting this view is a recent study of 12 patients with

advanced MM in which no deletions or mutations of either p15

or pitS genes were observed: however, hypermethylation of p16

and p/S were noted in 75 and 67% cases, respectively (34).

To directly test the biological significance of pitS gene

methylation, we treated malignant plasma cells with the de-

methylating agent 5-Aza-CdR. This agent both restored p16

protein and induced G1 growth arrest in patient PCL cells and

MM-derived cell lines, consistent with the view that p16 gene

methylation suppresses p16 gene transcription and thereby fa-

cilitates growth of these tumor cells. Because treatment with

5-Aza-CdR likely demethylates other genes besides p16 gene,

we further examined the effect of p16 protein on growth in

plasma cell dyscrasias by ectopically expressing p16 in MM cell




lines. Retrofection with pBabe-puro-p16 vector resulted in

sustained p 16 protein expression. Importantly, increased per-

centages of cells in G1 were present in those MM cell lines

transfected with pBabe-puro-p 16 vector compared to cells

transfected with control pBabe vector. This suppression of

growth in those tumor cells ectopically expressing p 16 protein is

the first demonstration that cell cycle regulatory proteins down-

stream of pl6, including CDK4/CDK6-pRB, are functional in

MM and further supports the view that loss of p16 contributes to

lack of growth control. On the other hand, our findings also

suggest that high levels of p16 protein, which are expressed in

MM cells, may regulate tumor cell growth, consistent with the

more indolent course of MM than of PCL. In addition, recent

reports demonstrate that ectopic expression of p16 not only

induces growth arrest, as in our study, but also may protect cells

from apoptosis (49, 50); we are currently examining whether

p16 protein expression similarly enhances survival of MM cells

or confers resistance to apoptosis induced by gamma irradiation

or dexamethasone.

Changes in tumor cell adhesion molecule profile, labeling

index, and interleukin 6 responsiveness are associated with

clinical progression of MM to PCL and its adverse prognosis

(51-53). The PCL patient samples and all MM cell lines in our

study were from patients with advanced stage disease. In this

setting, our findings demonstrate that decreased p16 protein

related to transcriptional inactivation of p16 gene by methyla-

tion occurs in PCL cells and likely also contributes to adverse

prognosis. This is analogous to those ALL patients whose tumor

cells lack p16 protein, in this case due to homozygous p16 gene

deletions, who have an adverse prognosis (15). A recent study

has also found hypermethylation of p16. often without alter-

ations of plS, to be much more frequent in cases of non-

Hodgkin’s lymphoma with high-grade than low-grade histology

(32). Within solid tumors, advanced stage tumors of brain, lung,

and head/neck have been reported to have higher rates of ho-

mozygous deletion of p16 gene (54, 55), suggesting that ac-

quired pl6 abnormalities may also contribute to disease pro-

gression in this setting. The finding of hypermethylation in PCL

in the current study, coupled with the association of hyper-

methylation of pl5/pl6 with blastic disease in MM (34), sug-

gests that p16 abnormalities may similarly play a role in disease

progression in MM. Therefore, when appropriate clinical spec-

imens become available. we intend to serially assess p16 cx-

pression in tumor cells from patients with MM who experience

progression to PCL.

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1997;3:2173-2179. Clin Cancer Res M Urashima, G Teoh, A Ogata, et al. and plasma cell leukemia.Characterization of p16(INK4A) expression in multiple myeloma

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