JOURNAL OF CELLULAR PHYSIOLOGY 135:133-138 (1988)

Expression of the CSF-1 Gene in the Blast Cells of Acute Myeloblastic Leukemia: Association With Reduced Growth Capacity

CHEN WANC, C.A. KELLEHER, G.Y.M. CHENC, JUN MIYAUCHI, CORDON G. WONC, STEVEN C. CLARK, MARK D. MINDEN, AND ERNEST A. McCULLOCH*

The Ontario Cancer Institute, Toronto, Ontario, Canada (C. W., C.A.K., G. Y. M.C., J, M., M. 0. M., E.A. M.); and the Genetics Institute, Cambridge, Massachusetts (G.G. W., S.C.C.)

Myelopoietic growth factors are known to influence t h e growth in culture of malignant blast cells from human Acute Myeloblastic Leukemia (AML). We have used cDNA clones for t h e factor CSF-1 and its receptor fms to study DNA and RNA from t h e blasts of 25 AML patients. The CSF-1 gene was always in t h e germline configuration. CSF-1 mRNA was found in about half t h e blast populations. The cells were also studied for their growth properties in culture. A highly significant association was found between CSF-1 expression and poor growth in suspension culture. Most blast populations expressed fms; t h e number of frns expression negative samples was too small to permit the detection of any association between fms expression and growth or any interaction between t h e effects of t h e expression of the growth factor and its receptor. We propose that CSF-1 may be an important part of the mechanism determining t h e balance between self-renewal and determination in AML blast clones.

I Hemopoietic stem cells may either renew themselves,

or alternatively, lose proliferative capacity while pass- ing through a limited number of terminal divisions as- sociated with differentiation (Siminovitch et al., 1963; McCulloch, 1983). It has been proposed that many can- cers, including leukemias, are malignant clones (Fi- alkow, 1976, 1982) maintained by the self-renewal of transformed stem cells (Selby et al., 1983; Buick and McCulloch, 1985). Studies in culture of the blast cells that form the predominant population in human Acute Myeloblastic Leukemia (AML) conform to this view (McCulloch, 1983); a minority of blasts are capable of colony-formation in culture (Buick et al., 1977). The blast colonies usually contain a few cells that produce colonies on replating (Buick et al., 1979); clonogenic cells may also increase exponentially in suspension culture, with a doubling time much longer than the generation time (Nara and McCulloch, 1985). Both observations support the proposal that clonogenic blast cells have the stem- cell property of self-renewal; blast cells are also capable of terminal divisions; often the end stage cells retain blast morphology. Sometimes they become plastic adher- ent develop processes, and have macrophage-monocytic cells markers (Langley et al., 1986). Such cells have not been observed to divide.

Growth in culture of blast stem cells usually depends on one or more of the growth factors first identified by their effects on normal myelopoiesis (Metcalf, 1986). Mo- lecular clones and recombinant proteins are available for several myelopietic growth factors capable of stimu- lating human cells in culture; these include: G-CSF (Ni- cola et al., 1985; Souza et al., 1986) which is normally restricted to the stimulation of granulopoiesis; CSF-1

0 1988 ALAN R. LISS, INC

(Wong et al., 1987), a potent stimulator of murine mac- rophage progenitors, but with less activity on human cells; GM-CSF (Gough et al., 1984; Miyatake et al., 1985) which leads to the production of granulocytes and mac- rophages and may also act on early cells with erythro- poietic potential; and IL-3 (Yang et al., 1986) which effects early multipotent stem cells.

We (Hoang et al., 1986; Kelleher e t al., 1987; Miyauchi e t al., 1987) and others (Griffin et al., 1986; Young and Griffin, 1986; Delwel et al., 1987) have demonstrated stimulation by these recombinant proteins of the growth in culture of AML blast stem cells. Considerable patient- to-patient variation was found in response. However, GM-CSF (Hoang et al., 1986), G-CSF (Kelleher et al., 19871, and IL-3 (Miyauchi e t al., 1987) usually stimula- ted both self-renewing and terminal divisions. In con- trast, we have observed recently that CSF-1 often favours terminal divisions, ending in macrophage-mono- cyte-like adherent blast cells incapable of growth (Mi- yauchi e t a]., 1988).

The availability of cDNA clones for growth factors made it possible to study the organization and expres- sion of the genes. Young et al. (1987) reported expression of GM-CSF genes in 11 of 22 AML blast populations. Six of these secreted growth factor, three of which formed colonies without added factor. We (Cheng et al., 1988) have reported altered configuration of GM-CSF or G- CSF genes in 4 of 20 AML patients and presented evi-

Received Aubwst 31, 1987; accepted December 4, 1987. *To whom reprint requests/correspondence should be addressed.

134 WANG ET AL.

TABLE 1. Expression of CSF-1, fms, and growth in culture of AML blast cells'

Cell Recovery Of Peripheral blood was obtained with informed consent Patient Diagnosis number clonogenic No. (FAB) CSF-l fms PE,, (106/m,) PE, cells(102) from 25 patients presenting at the Princess Margaret

Hospital or Sunnybrook Medical Centre. Patients were 1 M4 + + 17 0.6 10 selected for the study if sufficient blasts were present in

the blood for the cellular and molecular studies (see 2 M2 + + 74 1.7 94 3 M4 + - 1 1.7 0 4 M4 + + 0.5 0.6 39 23 below). The diagnosis of AML was made using standard 5 M1 + - 70 1.8 37 66 criteria; the FAB classification (Bennett et al., 1976) for 6 M4 + - 41 3 8.3 25 each is shown in Table 1.

Suspensions containing greater than 95% blast cells 7 M5 + + 112 0.4 6.5 2 8 M4 + + 2.5 1 21 21 9 M4 + - 15 0.7 20 14 and depleted of T-lymphocytes were obtained by two 10 M4 + + 400 4 4 16 centrifugations through ficol hypaque, the second fol- 11 M1 + + 0.2 0.8 2 '! lowing T-lymphocyte rosette-formation, as previously

described (Minden et al., 1979). The immunophenotypes 12 M4 + + o 1 0 13 M4 - + 160 2.2 184

- + 116 2.0 127 254 of the blast cells were obtained by indirect immunofluo- - + 120 1.6 145 232 rescence as previously described (Smith et al., 1983),

14 M4 15 M4 16 M4 - + 212 1.8 302 543 using My-1, My-7, My-9, OK-M1, M02, and HLA-DR. 17 M4 - + 24 0.6 95 57 18 M4 - + 2 1.3 44 57 Cell culture 19 M4 - + NA 0.7 518 362 20 M4 - - 0.4 2.2 5.7 13 The blasts were cultured using two previously de- 21 M5 - + 1 0.6 148 89 scribed methods. In both techniques supernatants from

the continuous bladder carcinoma cell line 5637 (5637- CM) (Buick et al., 1977; Hoang and McCulloch, 1985)

25 M1 - + 68 0.2 105 21 were supplied as a source of growth factors. These cells

mean offour replicate cultures with lo4 cells; Cell number: cell concentration in in our hands, 5637-CM almost always gives maximum

cultures with J c e I l s ; hcovery of clonogenic cells: The total clonogenic cells The first method, a clonogenic assay, depended on C O ~ -

(Linbro, Flow Lab, Mclean, VA) each well containing lo4 cells in a final volume of 0.1 ml medium consisting of alpha-MEM supplemented by 20% Fetal calf serum (FCS) (growth medium) and 5637-CM (10%). Colonies containing more than 20 cells were counted after 7 days of incubation in a moist atmosphere with 5% C02 (plat- dence that the changes could not be explained as restric- ing efficiency in methylcellulose or PEmc), Second, the

length polymorphisms* In three Of the cells were cultured at a concentration of lo6 celldm1 for four cases in which the structure of the gene was al- days in suspension in 35 mm lux petri dishes (Miles

for both G-CSF and GM-CSF was present' both Of the 5637-CM. Cells were harvested, counted, and plated in

neither GM-CSF nor G-CSF be detected in the plating efficiency of the cells from suspension (PEJ and supernatants of cultures of the blasts. In two instances, the cell count of the harvest (Nara and McCulloch, 1985). where the gene for GM-CSF was rearranged, abnor- mally large GM-CSF mRNA species were present; se- Southern blot analysis creted factor could not be detected and blasts from both DNA was extracted from the blast cells, digested with patients required the addition of factors for maximum Hind 111, Eco

Of mal!g- &ions), separated by agarose gel (0.8% agarox) electro- nant cell proliferation (Sporn and Roberts, 1985). While phoresis and transferred to nylon membrane (Gene

favours autocrine machanisms, the observation of ern, 1975). The membranes were hybridized with kb expression at the RNA level without detectable protein human CSF-l cDNA probe (Wong et al., 1987) labelled

tide primers and p2-dCTP, washed and autoradio- vation that some patients with factor expression still graphed as previously described (Cheng et al., 1986). require exogenous factor for growth.

In this paper we examine the organization and expres- sion of the gene encoding CSF-1 in AML blast cells. We of RNA and Northern blot analysis found that the gene was usually in the germline confg- uration. However, CSF-1 message was present in blasts Total cellular RNA was extracted from the leukemic from about 50% of patients. These CSF-1-message posi- blasts by the guanidine thiocyanate-caesium chloride tive populations were capable of much less growth in method (Chirgwin et al., 1979). Each RNA sample was suspension than negative populations. examined by electrophoresis on a 1.1% agarose gel and

MATERIALS AND METHODS Souce of leukemic cells

16:

404

E; 186

22 M4 - - 10 0.5 105 M4 - + 13 1.8 54 23

24 M4 - + 26 1 186

'PE,,: Plating efliciency of fresh blast cells in methylcellulosc; each value is the

suspension culture on day 7, from 1 x I06/mL on day 0; PE,: Plating efliciency of cells after 7 da s suspension culture, each value is the mean of four replicate

recovered from lo6 input cells in suspension culture, calculated from PE. and cell number; NA: not available.

are known to secrete a t least G-CSF, GM-CSF, and L-1;

growth, as be expected from its factor contents'

ony-formation in methylcellulose in 96 microwell plates

tered, dNA was found. In One instance, dNA Laboratories, Naperville, L), in growth medium and Same size as that detected in secreting The methylcellulose for colony-formation as described above.

Of this patient sew without added factor> The recovery of clonogenic cells was calculated from

one of the restriction enzymes growth in These data have been 'Onsidered in RI, or Pst 1 (according to the manufacturer's recornmen- light Of for the

the finding that Some patients active factors Screen plus, New England Nuclear, Boston, MA) (South-

requires ParticularlY in light Of the Obser- to a specific activity of 109 cp&pg by using o~igonu~ceo-

CSF-I GENE

1 2 3 4 5 6 7 8

2 8 s -

18s-

562 S

Fig. 1. Radioautographs of three Northern blots (1-3, 4-5, 6-8) after hybridization with a CSF-1 probe. The variation in intensity is evident. Lane 2 contains an example of blast RNA without evidence of CSF-1 expression.

stained with ethidium bromide to insure equal loading and no RNA degradation. 15 pg RNA samples were then fractionated on a 1.1% agarose gel with 6% formalde- hyde and transferred to a nylon membrane (Maniatis et al., 1982). The membrane was hybridized with the CSF- 1 probe or a probe for fms, a 1.4 kb pst I fragment of v- fms cDNA, kindly provided by Dr. C Sherr of St. Jude’s Childrens’ Cancer Research Hospital (Memphis, TN). Hybridization was performed in a solution containing 50% formamide, 1 molL NaCl, 1% sodium dodecyl sul- fate (SDS), 10% dextran sulfate and 100 pg, salmon sperm DNA in 42°C. The membrane was washed with 2 x SSC 1% SDS and 0.1 x SSC 1% SDS at 65°C for 2 h. Autoradiography was at -70°C using Kodak XARd film with intensifier screens. The membranes were then reprobed with hypoxanthine phosphoribosyltransferase gene probes, to insure that equal amounts of RNA were on the filters (data not shown).

Bioassay for CSF-1 Human CSF-1 activity was examined by using murine

bone marrow culture assay according to its effect on macrophage colony formation of murine cells. Recombi- nant human M-CSF was used as positive control.

RESULTS Northern analysis

RNA from blasts of the 25 AML patients was exam- ined for CSF-1 expression as described in “Materials and Methods.” Patient-to-patient variation was seen. In 13 instances message was not detected; in the remain- der, variable amounts of RNA were found that hybrid- ized to the CSF-1 probe. Figure 1 shows typical radioautographs with single 4 kb bands of different in- tensities. One example of a CSF-1 negative band is shown (lane 2, taken from a patient with FAB Md); the others are representative, including samples from pa- tients with FAB classifications MI, M4, M5.

The membranes were also hybridized with a probe from fms. Message was detected in all but 6 instances, although again quantitative patient-to-patient variation was seen. Figure 2 shows radioautographs with single bands, slightly larger than 4 kb.

EXPRESSION 135

Southern analysis We asked whether the failure to find CSF-1 message

could be explained by loss or re-arrangement of the gene. DNA from the 25 patients was examined by Southern blot analysis as described in the materials and methods. Representative autoradiographs are shown in Figure 3. The usual germline configuration was found with only one exception. In patient 11, Table 1 (lane 4, figure 3) 3 bands were seen with EcoR 1 digestion. The same pattern was found when DNA from remission bone marrow and EBV-transformed lymphocytes was probed. We conclude that the variation in expression of CSF-1 could not be explained by acquired changes in blast cell DNA; rather, the finding in patient 11 is a new example of restriction fragment length polymorphism.

Association of CSF-1 expression with blast growth characteristics

We asked whether the variable expression of CSF-1 was associated with the growth characteristics of the blasts population in culture. We used two complemen- tary methods; the clonogenic assay depends principally on terminal divisions while the suspension assay detects self-renewal (Nara et al., 1986; Wang and McCulloch, 1987). Data was collected on the plating efficiency of freshly-obtained blasts cultured in methylcellulose (PE,, as a measurement dominated by the effects of terminal divisions. The cells were cultured for 7 days in suspen- sion in the presence of 5637 CM, harvested, counted, and plated in methylcellulose. The plating efficiency before (PE,,) and after (PE,) suspension were not corre- lated (r = .280, ns) as expected, since PE, reflects self renewal events during suspension culture. The product of PE, and the cell count of the harvested culture gave the recovery of clonogenic cells after suspension, also a measure of blast stem cell renewal. The data is summa- rized in Table 1, together with the FAB status, CSF-1 and fms expression for each patient.

The most significant finding was an association be- tween CSF-1 expression and growth of clonogenic cells in suspension. The association was obvious when PE, and clonogenic cells recovery was compared for the CSF- 1 positive and CSF-1 negative patient subgroups. The relevant data are presented in Figure 4 as boxplots, a

2 8 S -

1 2 3 4 5

C-fms

18s - 893s

Fig. 2. A radioautograph of a Northern blot after hybridization with a v-fms probe. Variation in expression is evident.

136 WANG ET AL.

9.6 - 6.7-

4.0 -

2.5- Eco R I . {indm

Fig. 3. A radioautograph of a Southern blot after digestion with either Eco R1 or Hind 111 and hybridized with a CSF-1 probe. Lane 1 for both enzymes is a control using normal marrow. Lane 4, Eco R1 shows three bands, in contrast with the usual 2 band pattern. Studies with remis- sion marrow and EBV transformed lymphocytes provided evidence that this pattern is the result of restriction length polymorphism.

cl+ X A

I I I I I 1 I

0 50 100 150 200 250 300 ~ o ~ o n i e s ~ ~ ~ ' cells

f l X C

I 1 I I I I I 0 I 2 3 4 5 6(x104)

Recovery of clonogenic cells

x g , x E

-2 -I 0 I 2 3 4 ( x~04) Output clonogenic cells minus input clonogenic cells

Fig, 4. Boxplota showing the data for plating efliciency after suspen- sion culture PE, (A and B), recovery of clonogenic cells after suspension culture (C and I)) and change difference between input number of clonogenic cells and the number recovered after 7 days in suspension culture. Box plots are used commonly in exploratory data analysis to allow precise visual inspections of the forms of distributions. Each box contains the central 501 of the values in each group. The median is shown as (+) within the box. The lines extending to the right (high) or left (low) ends of the boxes extend to the largest observation within the 1.5 interquartile ran e of that end of the box, providing estimates of the skewness of the &tributions. Outliers are shown as asterisk (X). Boxes A, C, and E show the distributions of CSF-1 expression positive blasts; boxes B, D, and F, the distributions of CSF-1 negative blasts. The differences between the distributions are evident: A vs. B p = ,0002; C VS. D p = ,0016; E VS. F p = ,0017.

technique from exploratory data analysis that provides a visual display of distributions of data (Tukey, 1977). In these plots, the box represents the interquartile range, the median is shown as a cross and lines (whiskers) are extended to the left and right to show the most extreme value in the lower (left whisker) or upper (right whisker) quartile. Outliers are also identified. In addition, we calculated the change in clonogenic cell number in the suspensions from the day 0 input number to the day 7 number recovered. These data are also shown as box- plots in Figure 4. Each of the comparisons shows a highly significantly reduced growth in the CSF-1 posi- tive patients (p = .0002 for comparisons of PEs and p = .0016 for comparisons of clonogenic cell recoveries). The change in clonogenic cell number was calculated by subtracting the input clonogenic cells a t day 0 from the recovery a t day 7 (clonogenic cell output); the values were usually negative in the CSF-1 positive patients and positive in the CSF-1 negative group, a finding reflected in the p value (p = 0.0017) of the two groups. In contrast, no differences between the groups were found when PE,, was tested, indicating that the CSF-1 positive and negative patients had comparable plating efficiencies in methylcellulose before the suspension cul- tures were initiated and that this measure of terminal divisions was not associated with CSF-1 expression.

We also sought associations between growth charac- teristics and fms expression. There was a trend for fms positive cells to grow well. However, only 6 patients were fms expression negative, and no significant associ- ation was detected. Nor was a significant association found between immunologically-defined phenotype and CSF-1 expression or growth. Although many of the pa- tients belonged to FAB class Mq, patients with other FAB types were represented in both the CSF-1 expres- sion positive and negative groups and there was no association between FAB classification and CSF-1 expression or growth.

Comparison of expression in adherent and non-adherent blast cells

As noted earlier, blast cells that become adherent dur- ing suspension culture are terminal and unable to di- vide. In some patients, such adherent blasts are generated in suspension cultures in the presence of 5637- CM (Langley et al., 1986). We asked whether adherence was accompanied by a change in expression of fms or CSF-1. Blasts from three of the patients in the study produced enough adherent cells after a t least 2 weekly subcultures of suspension cultures to prepare RNA for analysis. Northern analysis was done on adherent and non-adherent cells, although half as much RNA from adherent cells compared to non-adherent was available for loading; the radioautographs of the RNA prepara- tions from patient 2, with the same membranes probed for CSF-1 and fms, are shown in Figure 5. It is apparent that a great increase was seen in CSF-1 and fms expres- sion; the change was greater for CSF-1 than for fms. The reduced amount of RNA from adherent cells loaded on the gels makes the differences in intensity more strik- ing. Similar results were obtained for blasts from pa- tient 12. Cells from patient 15 were CSF-1 expression negative before suspension culture and adherent cells did not become positive, although expression of fms in- creased (data not shown).

CSF- I

CSF-1 GENE EXPRESSION 137

like phenotypic characteristics in AML blasts, we are not prepared to propose that the CSF-1 expression ob- served reflects normal function surviving after transfor- mation. From this point of view, recent observations of Azoulay et al. (1987) may be relevent. They found that CSF-1, but not IL-3, GM-CSF, or G-CSF, was expressed during mouse embryogenesis. It may be, therefore, that CSF-1 expression in blasts may reflect embryonic behav- iour, a phenomenon often associated with malignancy.

The comparison of expression of CSF-1 and fms in adherent and nonadherent cells is consistent with this hypothesis. We examined populations that had been maintained long enough in culture to insure that few if any normal cells remained. The increase in fms in ad- herent blast cells was expected, since this population has other characteristics, such as increased expression of the antigen detected by the monoclonal M02 (Todd et al., 1981), associated with monocyte~macrophage differ- entiation. The increase in CSF-1 expression was Seen only in populations expressing the gene in non-adherent cells; but the observation is consistent with the associa- tion of expression of this factor with reduced growth in culture.

A 0 I 2 1 2

c - f mS

894s

Fig. 5. Autoradiographs of Northern blots made using Patient 2 RNA from non-adherent (lane 1) and adherent (lane 2) blasts cells, hybrid- ized to probes for CSF-1 (A) and for fms @). The gel for lane 2, (RNA from adherent blasts) was loaded with half as much RNA as the gels for lane 1 (nonadherent cells) since it was not possible to collect enough cells to obtain the full amount of RNA from the adherent populations. Thus, the increase in expression for both genes is very striking, partic- ularly for CSF-1.

Tests for CSF-1 secretion by blast cells Supernatants from cultures of blast cells were tested

for CSF-1 biological activity, using mouse marrow cells as targets. Positive effects were not found, regardless of whether or not the blast cells were expressing CSF-1 d N A . Mixing experiments provided no evidence of secretion of CSF-1 inhibitors by blast cells.

DISCUSSION In this paper we extend our studies of gene organiza-

tion and expression in AML to CSF-1 and its receptor, fms (Sherr et al., 1985). Using the several restriction enzymes, we found the CSF-1 gene to be in the germline configuration in 24 of 25 instances, including one sam- ple where an abnormality was seen in the configuration of the GM-CSF gene (Cheng et al., 1988). In the remain- ing case, an unusual pattern was detected with Eco R1, that could be explained as a novel polymorphism. In contrast, expression was not uniform; about half the blast populations were positive for CSF-1 d N A while the remainder were negative. Nineteen of the 25 speci- mens contained fms mRNA and only six did not.

The variation in expression of CSF-1, with an almost even distribution between positive and negative blast clones, led to the most striking observation of the paper; a highly significant association was found between CSF- 1 expression and poor growth of clonogenic cells in sus- pension culture. In contrast, there was no association between CSF-1 expression and initial plating efficiency. We consider that these results indicate that CSF-1 expression is one of the attributes that determine the balance between self-renewal and differentiation in AML blast cells.

In normal adult hemopoiesis CSF-1 is secreted by monocytes and other mesencymal cells; it is usually considered to stimulate macrophage presurors and their progeny (Metcalf, 1984). However, since there was no association between CSF-1 expression and macrophage-

As in our experience with cells expressing GM-CSF (Cheng et al., 1987), we were unable to find CSF-1 activ- ity in supernatants of cultures of expression-positive blasts. Thus, our data with CSF-1 do not contribute directly to a test of an autocrine hypothesis. However, in other experiments (Miyauchi et al., 1988) we found the addition of rCSF-1 to AML blast cultures often led to increases in adherent cells and a reduction in self- renewal (Miyauchi et al., 1988). It seems possible, there- fore, that the CSF-1 message is translated, but more sensitive methods will be required to detect the protein in either an extracellular or intracellular location.

The distribution of fms expression among the AML clones in this study, with its low number of expression- negative examples, did not disclose a significant rela- tionship between fms expression and growth; nor was it feasible to use the data to uncover interactions between fms and CSF-1.

Finally, it would be important to determine whether or not the reduced growth capacity seen in the CSF-1 expression positive group was associated with a good clinical prognosis. Since the study population was se- lected on the basis of high peripheral blast counts, the rate of successful remission induction was much less than that encountered in unselected patients; therefore the sample was not suitable for analysis of clinical out- come. Regardless, the findings may contribute to our understanding of the basis of heterogeneity in AML and support the view that CSF-1 may be important in the regulation of the growth of leukemic stem cells.

ACKNOWLEDGMENTS This study was supported by grants from the Medical

Research Council of Canada and the National Cancer Institute of Canada. J . Miyauchi was supported by the Fukuzawa Memorial Fund from Keio University, Tokyo, Japan. M.D. Minden is a scholar of the Leukemia Soci- ety of America.

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