Proliferation, Differentiation Arrest, and Survival in Leukemic Blast Cells" S. FERRARI) R. MANFREDINI, A. GRANDE, G. TORELLI, AND U. TORELLI ExperimentalHematology Center II Medical Clinic University of Modena via Del Pouo 71 Policlinico 41100 Modena, Italy CELL CYCLE CHARACTERISTICS OF LEUKEMIC BLAST CELLS

Years ago, a study of parameters such as labeling index, mitotic index, and determination of DNA content showed that the growth fraction (i.e., the pool of cycling cells) is very low in most acute leukemia blast cell populations, suggesting that most leukemic cells do not actively proliferate.' The normal bone marrow demonstrates a dynamic balance between quiescent, cycling, and differentiating cells, and the proportion of cells in each pool remains the same under physiological conditions.* Thus, hematopoietic cells provide an excellent experimental system for molecular analysis of the control of cell proliferation and differentiation. In acute leukemia the balance between the various pools is different. Study of the origin of such differences can explain why leukemic blast cells acquire different degrees of growth advantage over their normal counterparts. The growth advantage is due in part to increased survival of leukemic blast c e k 3 In fact, these cells leave the pool of proliferating cells without progressing towards terminal differentiation. This maturation arrest of leukemic blast cells explains the increased white cell count in the bone marrow as well as the peripheral blood of the majority of patients with acute leukemia. In the last few years, several groups have used various approaches to isolate cell-cycle related genes in different eukaryotic cells. These approaches include differential screening of cDNA libraries from poly(A)+RNA derived from serumstimulated cells4 and, more recently, the use of subtraction cDNA libraries5 or the correction, by means of DNA transfection, of the defect in temperature-sensitive mutant cells lines of the cell cycle.6 Furthermore, the mRNAs of several oncogenes, such as c-fos, c-myc, c-myb, and h-ras, are serum or growth factors regulated in different cell systems such as serum-stimulated quiescent fibroblasts or peripheral blood lymphocytes stimulated with mitogens.' These oncogenes are usually expressed during the G1 phase of the cell cycle.8 The mRNA of other genes, such as histone H3, thymidine kinase, and thymidine synthase, is induced during the S phase of the cell cycle. With these molecular markers of the G1 and S phases of the cell cycle, and with the likely identification of genes associated with the G2 and M phases aThis work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.). 'Address for reprint requests: Dr. Sergio Ferrari, Experimental Hematology Center, Second Medical Clinic, University of Modena, Via Del Pozzo 71, Policlinico, 41 100 Modena, Italy. 202

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of the cell cycle, it will be possible to carefully monitor the proliferative state of different cell populations. This panel of molecular markers may allow molecular monitoring of the cell cycle similar to way monoclonal antibodies are used for immun~phenotyping.~ We have demonstrated a high correlation between the levels of expression of histone H3 mRNA, specifically expressed in the boundary G1-S phases of the cell cycle, and the mitotic index.’” Consequently, the abundance of histone H3 mRNA is a good indicator of the growth fraction, that is, the number of cycling cells in the cell population studied. Furthermore, we showed that in asynchronous proliferating systems, G1 and S phase-related genes are fairly well expressed, and the ratios of expression between G1 and S specific genes are fairly constant.” With Northern blot analysis we studied the levels of expression of these G1 and S related genes and oncogenes in several blast cell populations of AML and ALL. Our purpose was to compare the levels of expression of GI and S specific genes in order to characterize the majority of blast cells of leukemic populations. The most commonly available techniques, such as flow cytornetry, cannot distinguish cells in GO or G1 phases of the cell cycle. With Northern blot analysis we showed a differential expression of G1-related genes in acute leukemia blast cells (FIG. 1). Synthesis of the data shows that in 50% of the cases of acute leukemia studied, middle G1 genes such as vimentin and calcyclin are highly expressed, in 25% of cases c-myc is mainly expressed, in 10% of cases only histone H3mRNA is detectable, and in the other 10% of cases all genes are clearly detectable. We postulate that at least in part of the blast cell population, these cells lose the capacity to progress through the G1 phase of the cell cycle and become arrested at different stages of the G1 progression, the early stage (cells capable of expressing, for example, the c-myc gene), middle stage (cells capable of expressing genes such as vimentin and calcyclin), and middle-late stage (cells capable of expressing p53 or ODC genes).I2 Progression through the cycle of leukemic blast cells mainly expressing the histone H3 gene may depend on a very short GI progression phase. Indeed, fibroblasts infected with the adenovirus or SV40 enter the S phase of the cell cycle without growing in size and without expressing early G1 associated genes.I3 The small percentage of leukemic cells capable of expressing all the G1 and S associated genes may be due to heterogeneous blocking of the cell cycle progression. We consider the inability of leukemic blast cells to progress through the cell cycle to be a kind of “pathology of the cell cycle.” The underlying hypothesis is that the leukemic blast cells progressively lose the ability to proliferate and consequently stop mainly in the G1 phase of the cell cycle (as already discussed). To support this hypothesis we studied the expression of several cell cycle genes at different stages in the reverse progression from S to GO in WI-38 human fibroblasts. The abundance of histone H3, ODC, vimentin, calcyclin, ADP/ATP translocase, c-myc, and p53 mRNAs decreases progressively, each mRNA species disappearing with different kinetics during the induction of quiescence in WI-38 fibroblasts (FIG. 2 ) . The pattern is essentially opposite that detected in WI-38 stimulated with serum or purified growth factors. Among the genes that we studied, the levels of c-myc and p53 mRNAs appear to be critical. In experiments in which only platelet-poor plasma (PPP; plasma without PDGF) was used to stimulate WI-38 fibroblasts, only the cells still expressing c-myc and p53 mRNAs were able to progress to the S phase, whereas when the mRNAs of these two genes were no longer detected, the cells failed to respond with DNA synthesis to PPP s t i r n ~ l a t i o nThe . ~ ~molecular data on cell cycle gene expression are in keeping with several previous reports on the macromolecular metabolism of ribosomal and heterogeneous nuclear RNA. Several years ago we showed that in most leukemic blast cell populations the rate of processing ribosomal as well as messenger RNA is extremely low, leading to the accumulation of

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unprocessed tran~cripts.’~J~ These studies, together with the study of the complexity and abundance of polyadenylated RNA sequences of quiescent, proliferating, and terminally differentiated cells of both normal and leukemic blast cell populations, clearly demonstrated that acute leukemic blast cells cannot be considered as quiescent just as they cannot be regarded as proliferating or terminally differentiated c e l l ~ . ’ ~These J ~ data fully support the hypothesis that acute leukemia blast cells progressively lose the ability to proliferate and arrest mainly in the G1 phase of the cell cycle. They also are unable to progress through the differentiation pathway, never spontaneously reaching terminal differentiation. All data described in this section support the evidence that leukemic cell populations are extremely heterogeneous in their biological behavior. To conclude this section, it is likely that study of the expression of cell cycle genes and oncogenes in acute leukemia blast cells, before providing new clues as to what leads the cells to “uncontrolled proliferation,” will help to elucidate what leads cells

1.5 kb-

ADPIrn vend.

a7kb-

calcyclh

2.4 kb-

C - W

19kb-

a5 kb-

22kb-

-

vimnth

H3

I)-Actin

123456789lO11l2X3141516 FIGURE 1. Composite picture of the autoradiographs from Northern blot analysis of total cellular RNA isolated from different cell lines and from different cases of acute leukemia blast cell populations indicating the levels of expression of several cell-cycle related genes that are labeled on the right side of the picture. The source of RNAs in the various lanes is as follows: lane I, quiescent WI-38 human fibroblasts; lane 2, proliferating WI-38 human fibroblasts; lane 3, a Burkitt lymphoma cell line (Daudi); lane 4, proliferating HL-60 cells (a human myeloid leukemic cell line); lanes 5-16, different leukemic blast cell populations with different phenotypes. The molecular probes used in this study as well as the Northern blot procedure are already d e s ~ r i b e d . ’ ~ * ~ ~ . ~ ~

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2,4 kb-

c-myc

2,2 kb-

ODC

1,9 kb-

Vimentin

0,5 kb-

Hlstone HI

FIGURE 2. Composite picture of the autoradiographs from Northern blot analysis of total cellular RNA isolated from WI-38 human fibroblasts. The RNA was extracted from these cells at different times (day 2 to day 20) of quiescence (confluent cells 0.5% fetal calf serum). After 20 days of quiescence the cells were stimulated to proliferate with 15% fetal calf serum. The molecular probes used and the Northern blot procedure are already described.14

+

with a neoplastic phenotype to a condition of proliferation arrest. We do not know at present to what extent inhibition of the progression through the cell cycle is due to mechanisms intrinsic to leukemic cells or to peculiar aspects of the microenvironment such as the abnormal production of growth factor^.^^-^' POSTTRANSCRIPTIONAL ALTERATIONS IN GENE EXPRESSION PRESUMABLY ORIGINATE THE GROWTH ARREST OF LEUKEMIC CELLS Gene expression is regulated at different levels during the events that occur from transcription of the template to formation of an adequate amount of functional A critical process in this cascade is the coordination of three essential protein.22*23 steps: (1) Processing of nuclear RNA with formation of mature mRNA24925; ( 2 ) cleavage of ribosomal precursor RNA26;and (3) assembly of proteins in functional 40s and 60s ribosomal particles.27 The coordination of such molecular events appears particularly complex because the involved genes are located on many different chromosome^.^^^^^ The increased formation of new ribosomes, which occurs during the G1 progression phase of the cell cycle, is a complex process involving the

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coordinate synthesis and processing of rRNA and the synthesis of the respective ribosomal protein^.^^^^^ The events that regulate this process are still poorly understood. In mouse fibroblasts, the increased abundance of rRNA is due to an increased rate of transcription and a high rate of processing rRNA precursor^.^^ The increase in ribosomal proteins in the same cells is the result of increased efficiency of translation of rpmRNA due to a shift in the association with polysomes rather than to an increase in the transcription rate.32 These two components of ribosomes, however, must be correlated, because inhibition of ribosomal protein synthesis inhibits the processing of ribosomal precursor^.^^,^^ The steady-state levels of mRNAs coding for ribosomal proteins S6, S11, and S14 were evaluated in quiescent and proliferating human fibroblasts and in resting and proliferating human peripheral blood mononuclear cells. Expression of the rp genes studied appears to be constitutive and coordinately regulated. In 15 different leukemic populations studied, at variance with what we observed in normal cells, the abundance of each rpmRNA is remarkably different from the other. Therefore, the results of our experiments indicate that expression of the three ribosomal protein genes that we studied undergoes independent, noncoordinated changes in the majority of the leukemic populations studied. In these populations the abundance of each rpmRNA is different from that in the others.35The coordinated regulation of ribosomal proteins synthesis in these cells is impaired, making it difficult for all the events that depend on proper assembling of ribosomes to occur. These events include the processing of rRNA precur~ors,3~ which is apparently deeply involved in the disorder, leading to the arrest of the proliferation of leukemic blast cells. Time course studies carried out in our laboratory several years ago had shown that the rate of processing of primary transcript is abnormally low in leukemic blast cells, leading to the accumulation of unprocessed transcript^.^^ The same studies gave clear evidence of the inability of leukemic cells to produce mature 18s ribosomal RNA.37Recent studies38also have confirmed the previous assessment that impaired processing leads to the accumulation of 32s ribosomal RNA.38 These experiments present solid evidence that posttranscriptional abnormalities characterize the leukemic cells. Many other results support this important conclusion. Solid evidence obtained in time course experiments and recently by direct assay of the primary transcript indicates that so-called nuclear RNA accumulates in leukemic cells.38Thus, we have additional evidence of posttranscriptional peculiarities in leukemic leukocytes. Still other experiments carried out in our laboratory confirm the importance of posttranscriptional alterations of gene expression in leukemic blast cells. In fact, we used Northern blot and Western blot analysis to study the levels of expression at mRNA and protein levels of two growth-related genes such as vimentin and c-rnyc. Our results show that the most remarkable event in these cell populations is the accumulation of mature mRNA molecules.39This accumulation, in the absence of a proportionally increased protein synthesis, may be the result of a peculiar compartmentalization of mRNA molecules, which cannot be efficiently translated, or of an inefficient translational apparatus as already mentioned. In other leukemic cell populations we found only the expression of mature mRNA and not that of the corresponding protein.40 Several alterations in the regulation of gene expression at the posttranscriptional or translational level may be considered as the origin of proliferation arrest and, as we are going to show, of the differentiation arrest occurring in leukemic blast cells. DIFFERENTIATION ARREST OF LEUKEMIC CELLS

It is commonly accepted that leukemic blast cells are arrested at different stages along the differentiation pathway and never spontaneously reach terminal differenti-

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ation. We do not yet know the correlation between proliferation and differentiation despite an incredible amount of experimental work on the kinetics of these cells4‘ and on the mechanisms of action of growth factors and their receptor^.^^,^^ In fact, several cytokines are capable of triggering the target cells simultaneously along both the proliferative and the differentiative pathways. However, in vitro cultured leukemic blast cells can be rescued only to proliferate when incubated with purified growth f a ~ t o r s .On ~ ~the , ~ other ~ hand, acute promyelocytic leukemic blast cells carrying the

Go 1

GO

G1

‘

G1

‘&

FIGURE 3. Schematic representation of the cell cycle characteristics of hematopoietic cells in different functional states. In the upper scheme is indicated a quiescent cell (GO) that can be triggered in the GI phase of the cycle by the action of growth factors. In this situation the G I phase of the cycle is particularly prolonged, and several cell cycle-related genes are activated. This pattern can be attributed to the quiescent hematopoietic stem cells and is actually observed in peripheral blood lymphocytes when they are recruited in cycle. In the middle scheme is indicated a cycling cell. This pattern may be regarded as proper for a population of self-mantaining stem cells or immortalized cells in culture. The lower scheme indicates the characteristic of the cell cycle of differentiating cells. In these cells, proliferation and differentiation occur simultaneously, but a progressive increase in the length of the GI and S phases is evident. Furthermore, these cells stop mainly in the G1 phase of the cycle and the differentiation process becomes irreversible. This pattern may be demonstrated in proliferating and differentiating committed cells of the bone marrow.

t(15;17) in which the retinoic acid receptor (alfa chain) is juxtaposed on the Mil gene4’ are capable of differentiating in vivo into granulocytes when treated with all-trans-retinoic acid.48When the differentiation commitment of a normal precursor cell occurs, the G1 phase of the cell cycle progressively increases in length, and terminally differentiated cells are practically arrested in this phase of the cycle and cannot be triggered along the proliferative pathway (irreversible specialization) (FIG.3). Furthermore, previous studies have shown that high levels of expression of genes involved in the proliferative pathway can inhibit cell differentiation. The effect of these genes on terminal differentiation is due not to significant alterations in normal growth control mechanisms but instead to an inability of cells to commit to a

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pathway leading to the terminally differentiated state. In fact, the genetic program of terminal differentiation often, if not always, requires the permanent withdrawal of several cell-cycle gene-related products. We can include among these genes c-myc, c-myb, erb-A, c-jun, and c-fos. For example, mouse erythroleukemia cells expressing a recombinant c-myc gene fail to other examples are the suppression of erythroid differentiation by overexpression of the c-myb o n c ~ g e n e and ~ ~ suppres-~~ sion of myogenesis by the increased expression of erb-B.55Overexpression of c-jun can block the differentiation pathway in Friend erythroleukemia cells induced by DMS0.56The preliminary conclusion of these experiments is that several oncogenes are involved primarily in cell proliferation and that cell differentiation requires permanent withdrawal of these gene products and the permanent exit from the cell cycie57(TABLE1). To approach the problem of the possible correlation between the proliferative potential and the differentiation capacity, we studied the capacity of HL-60 myeloid cells (M2 type)ss,59to differentiate after complete inhibition of proliferative activity. We treated these cells with antisense oligodeoxynucleotide (AS aODN) specific for TABLE1. Effects of Increased Gene Expression on Cell Proliferation and Differentiation Gene c-mvc c-myb

Cells F-MELC 3T3/L1 preadipocytes HL-60 SKT-6 F-MELC HL-60

jun jun-B jun-D

F-MELC

C-~OS

SKT-6

Effects Inhibition of cell proliferation and differentiation of Friend erythroleukemia cells, preadipocytes, and myeloid HL-60 cells Inhibition of cell differentiation of murine erythroleukemia cells induced by DMSO; inhibition of granulocytic differentiation of HL-60 cells induced with R.A.-DMSO Inhibition of Friend erythroleukemia cell differentiation induced by DMSO Inhibition of erythroid differentiation of Friend leukemia cells induced by erythropoietin or chemical inducers

the mRNA of the c-myb oncogene which was shown to be a regulating gene for the proliferative pathway. In fact, this antisense c-my6 oligodeoxynucleotide can inhibit the proliferation of several cell lines and human normal hematopoietic progenitors.60,h1After 5 days of this treatment the HL-60 cells were arrested mainly in the G1 phase of the cell cycle (FIG.4). G1-arrested HL-60cells were induced to differentiate with several specific inducers such as retinoic acid62or DMS063for the granulocytic pathway; phorbol esters (TPA)64for macrophage differentiation and with diidrocalciferol (vitamin D3) for the monocytic differentiation.6s Our results clearly show that G1-arrested HL-60 cells lose the capability to differentiate along the granulocytic pathway when treated with retinoic acid or DMSO, but they retain the capability to differentiate along the monocytic pathways only. When these cells are induced with TPA or vitamin D3 to macrophage and monocytic differentiation, respectively, these pathways are normally activated.66We can conclude that granulocytic differentiation requires the cell proliferation c-myb dependent leading to the hypothesis that in this differentiation pathway, proliferation and differentiation must proceed simultaneously. This does not occur in monocytic or macrophage differentiation pathways

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0

0

30

60

PI.

FIGURE 4. Flow cytometric estimation of DNA fluorescence intensity of HL-60 cells treated for 120 hours with c-myb antisense (left) or sense (right) oligomer. In each panel, the rectangles marked 1, 2, and 3 represent the cells in the GO/Gl, S, and G2/M phases of the cell cycle, respectively. The technique has already been described.h6

which can normally be triggered in cells whose proliferative capacity is blocked (FIG.5 ) . Knowledge of the mechanisms that control cell differentiation is still primitive. A promising approach is the selective and specific inhibition of the expression of a gene product required for normal differentiation. This leads to an understanding of the possible biological function of the gene studied in the differentiation process (TABLE 2). Particularly interesting are the results obtained with the AS aODN strategy6’sa to inactivate the product of two oncogenes in myeloid cell differentiation. The first oncogene is thejins. It was identified as the transforming gene of the McDonough granulocytic and mono-macrophagic differentiation

k

quiescent cells (GO) transition

monocytic rnacrophagic differentiation

’ and

FIGURE 5. Schematic representation of the cell cycle showing the possible correlation between proliferation and differentiation. It is particularly interesting that monocytic and macrophage differentiation can be triggered in myeloid HL-60 cells starting from the G1 phase of the cycle, whereas granulocytic differentiation can be triggered only in cells that are capable of proliferating activity.

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strain of the feline sarcoma virus which also transforms fibroblasts. The cellular homolog for fms is the receptor for CSF-1, which is a factor involved in the differentiation and perhaps proliferation of monocytes and macrophages. Inactivation of the c-fms mRNA completely inhibits the capacity of HL-60 cells to differentiate along the monocytic pathway.69 Another oncogene that we studied, which is related to granulocytic differentiation, is the c-fes, which is the cellular homolog of the fpslfes transforming gene of the feline sarcoma virus. cfes is expressed only in myeloid cells, and its mRNA abundance increases several times in HL-60 myeloid cells induced to differentiate along the granulocytic pathway, its highest level of expression having been observed in normal human bone marrow g r a n ~ l o c y t e . ~ ~ ~ ~ ~ Inactivation of the c-fes mRNA with a specific antisense oligodeoxynucleotide in HL-60 myeloid cells (M2 phenotype) does not interfere with the proliferating capacity of these cells. It does, however, completely inhibit the granulocytic terminal differentiation inducible with retinoic acid or DMSO because of progressive cell death. The same AS aODN does not interfere with monocytic differentiation TABLE2. Biological Effects of Single Gene Inhibition on Cell Differentiation Target Gene

Cells

Biological Effects

c-fes

HL-60

c-fms

HL-60

Protein kinase, type I1 beta

HL-60

MERS

MEL

Myogenin

L6-A1

Cell proliferative activity is unaffected; complete inhibition of granulocytic differentiation with cell death; macrophage adhesion is lost while monocytic differentiation is unaffected Inhibition of macrophage differentiation induced by PMA or GM-CSF without any interference with the granulocytic differentiation induced by DMSO or monocytic differentiation induced by vitamin D3 Reduction of cyclic AMP levels followed by inhibition of the proliferation and differentiation capacity with the exception of the differentiation pathway induced by phorbol ester Inhibition of erythroid differentiation induced by DMSO Inhibition of muscular differentiation induced by the growth factor IGF-1

inducible by diidrocalciferol (vitamin D3) and only marginally interferes with the macrophage differentiation pathway inducible in these cells by phorbol esters (TPA). It is worth mentioning that inhibition of HL-60 cell proliferation with a specific c-myc AS aODN leads to spontaneous differentiation of these cells along the monocytic path~ay.~~.~~ PROLONGED SURVIVAL AND AGING AS A MAIN FUNCTIONAL CHARACTERISTIC OF LEUKEMIC CELLS We thus arrived at certain conclusions regarding the leukemic blast cell: 1. These cells can no longer be considered “highly proliferating cells”; rather, proliferative activity is confined to a small fraction of the entire population, the rest consisting of cells that biologically are difficult to define. 2. The rate of proliferation as documented by all indices is definitively low, although some cells can be reinduced to proliferate under particular conditions. 3. The molecular biology of cell cycle genes has yielded convincing evidence that

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these cells are arrested in the G1 phase of the cycle at either the beginning, the middle, or the end of this phase. Therefore, these cells are blocked before reaching the S phase but in no way can be considered resting cells. 4. Molecular biology also gives a clue to the origin of this arrest. Apparently several posttranscriptional disorders of the complex pathway regulating gene expression lead to the accumulation of unprocessed or partially processed products of the original transcript, inhibiting its functional expression. 5. Inhibition of the proliferation of these cells is related to inhibition of differentiation. The latter phenomenon, which has been known a long time, is definitely related to inhibition of proliferation; however, the relation between these two events is still poorly understood. Unquestionably, some cell cycle-related genes are also differentiation related; a typical example is represented by c-myb whose expression is necessary for both myeloid proliferation and differentiation. Much work is still needed, however, to understand the biological events. Their complexity is exemplified by the ill-defined function of another oncogene necessary for myeloid differentiation, c-fes. In fact, inhibition of this gene inhibits granulocytic maturation, leading to precocious death of myeloid precursors presumably by programmed cell death. In conclusion, if we try to define a leukemic blast cell in terms of average life span and cell survival, we have to conclude that these leukemic cells, because of the posttranscriptional defects that seem to characterize them, are endowed with a reduced rate of senescence and an increased life span, which is one of the main factors in their lethal effect on the organism. REFERENCES 1. LAERUM, 0. D. & T. FARSUND. 1981. Clinical application of flow cytometry: A review. Cytometry 2: 1-13. S. & R. BASERGA. 1987. Oncogenes and cell cycle genes. BioEssays 7: 9-13. 2. FERRARI, 3. HOEUER,D., E. B. HARRIS,T. M. FLIDNER & H. HEIMPEL. 1972. The turnover of blast

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cells in peripheral blood after in vitro 3H-Cytidine labeling and retrotransfusion in human acute leukemia. Eur. J. Clin. Invest. 2: 259-261. HIRSCHHORN, R. R., P. ALLER,Z . YOUAN, C. W. GIBSON & R. BASERGA. 1984. Cell-cyclespecific cDNAs from mammalian cells temperature sensitive for growth. Proc. Natl. Acad. Sci. USA 81: 6004-6008. SCHNEIDER, C., R. M. KING& L. PHILIPSON. 1988. Genes specifically expressed at growth arrest of mammalian cells. Cell 5 4 787-793. GRECO,A,, M. ITTMAN & C. BASILICO. 1987. Molecular cloning of a gene that is necessary for G1 progression in mammalian cells. Proc. Natl. Acad. Sci. USA 8 4 1565-1569. DENHARDT, D. T., D. R. EDWARDS & C. L. J. PARFETT. 1986. Gene expression during the mammalian cell cycle. Bichim. Biophys. Acta 865: 83-125. REED,J. C., J. D. ALPERS,P. C. NOWELL & R. G. HOOVER. 1986. Sequential expression of protooncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. Proc. Natl. Acad. Sci. USA 8 3 3982-3986. FOON,K. A. & R. F. TODD,111. 1986. Immunologic classification of leukemia and lymphoma. Blood 6 8 1-31. FERRARI, S., F. NARNI,W. MARS,L. KACZMAREK, D. VENTURELLI, B. ANDERSON & B. CALABRETTA. 1986. Expression of growth-regulated genes in human acute leukemias. Cancer Res. 4 6 5162-5166. FERRARI, S., B. CALABRETTA, L. SELLER], G. CECCHERELLI, G. TORELLI & U. TORELLI. 1988. Expression of oncogenes and cell cycle related genes in acute and chronic leukemias. Leukemia 2 160-166. TORELLI, U. & S. FERRARI. 1987. Oncogenes and cell cycle genes and proliferation arrest in acute myeloid leukemia. In Genotypic, Phenotypic and Functional Aspects of Haematopoiesis. F. Grignani, F. M. Martelli, & D. Y. Mason, eds Vol. 41: 123-131. Serono Symposia Publications. Raven Press. New York, NY. LIU,H. T., R. BASERGA & W. E. MERCER.1985. Adenovirus type 2 activates cell-cycle

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