Inactivation of the retinoblastoma gene in human ...

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1991 77: 833-840

Inactivation of the retinoblastoma gene in human lymphoid neoplasms AM Ginsberg, M Raffeld and J Cossman

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Inactivation of the Retinoblastoma Gene in Human Lymphoid Neoplasms By Ann M. Ginsberg, Mark Raffeld, and Jeffrey Cossman The absence of wild type retinoblastoma (Rb) gene expression in a wide variety of human solid tumors suggests an etiologic role for this tumor suppressor gene in human cancer. We have evaluated the involvement of Rb gene inactivation in the pathogenesis and progression of human lymphoma and leukemia.We examined the genomic configuration and transcription of the Rb gene in cultured cell lines and primary cases of T- and B-cell lymphomas and leukemias. By Southern analysis, abnormalities of the Rb locus were identified in 1 of 5 T-cell acute lymphoblastic lymphoma (T-ALL) cell lines, 1 of 26 primary cases of T-ALL, 1 of 40 primary cases of chronic lymphocytic lymphoma/well-

differentiated lymphoma (CLL/WDL), and 1 of 15 primary cases of intermediately differentiated lymphoma (IDL). By Northern analysis, markedly reduced or abnormal expression of the Rb gene was identified in 2 of 5 T-ALL cell lines, 1 of 7 primary cases of T-ALL, 1 of 5 primary cases of CLL/WDL, and 1 of 6 primary cases of IDL. These findings show that Rb gene inactivation can be associated with a broad range of lymphoid neoplasms and that loss of the tumor suppressor function of Rb may influence the pathogenesis and progression of lymphoma/leukemia. This is a US government work. There are no restrictions on its use.

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Culture Collection [ATCC], Rockville, MD), MOLT-4 (ATCC), HE HYPOTHESIS THAT inactivation of tumor supJurkat (ATCC), CEM (ATCC), and 8402.” Epstein-Barr viruspressor genes contributes to carcinogenesis stems transformed B-lymphoid lines SB (ATCC) and 8392” were previfrom the two-event model fostered by Knudsen, based on ously derived from the same individuals as the HSB-2 and 8402 his observations of inheritable susceptibility to retinoblasT-ALL lines, respectively. Peripheral blood lymphocytes (PBL) toma (Rb).’ Genetic linkage analysis localized the Rb were obtained by Ficoll-Hypaque density-gradient centrifugation susceptibility gene to chromosome 13q14.2-5The Rb gene of normal human peripheral blood. encodes a nuclear phosphoprotein whose phosphorylation DNA and RNA preparations. High molecular weight, genomic is regulated in a cell-cycle-specific manner.”9 Inactivation DNA was extracted from cell lines growing in suspension culture of the Rb gene has been implicated in the pathogenesis of a and from tissue samples according to standard technique^^^^^^ and variety of human solid tumors, including osteosarcoma and quantified by absorbance measurements. Integrity of DNA samsoft tissue sarcoma^^""'^ and carcinomas of the brea~t,’~.’~ ples was determined by gel electrophoresis. Total cellular RNA was extracted from cells growing in suspension, and from frozen and bladder? as well as Rb. Although cancer cells and tissue samples stored under liquid nitrogen using RNAdevelopment is a multifactorial process, these findings 201, according to the manufacturer’s instructions (CinnaiBiotecx suggest that common genetic abnormalities occur in many Laboratories International, Inc, Friendswood, TX). Integrity of different cancer types and may help explain the deregulated RNA samples was determined by formaldehyde-agarose gel electrogrowth control associated with malignancy. phoresis. Lymphoid neoplasms represent a broad variety of disease cDNA probes. cDNA probes, designated Rb0.9 and Rb3.8 and states composed of clonally expanded populations of T or B representing 5’ and 3’ portions of the Rb cDNA, respectively, were lymphocytes. The discovery of activation of genes, particuthe generous gifts of Dr Stephen Friend (Boston, MA) and are larly as a consequence of chromosomal translocation, in depicted in Fig 1A (see also reference 4). Chicken p-tubulinzs and actinz6probes were the kind gifts of Dr Andrew Larner (Bethesda, lymphoid neoplasms has focused attention on the role of MD). Probes were labeled with (”P) by the random primer oncogene products and other growth-promoting cellular method” using the Bethesda Research Laboratories (BRL) Ranconstituents in the pathogenesis of these diseases. Howdom Primers DNA Labeling System (BRL, Life Technologies, Inc, ever, little information exists regarding the possible role of Gaithersburg, MD) essentially according to the manufacturer’s inactivation of tumor-suppressor genes in lymphoma or directions, except that additional . ”P-dCTP was sometimes leukemia. To extend the observations of Rb in solid tumors, added to increase probe specific activity. Probes were labeled to we used DNA and RNA blot hybridization analyses to specific activities of 1 to 3 x io9cpm/pg. survey primary tissues and cell lines of T- and B-cell Southem blots. Genomic DNA samples (10 to 15 pg) were neoplasia for derangements of the Rb gene. Using these digested with restriction endonucleases, HindIII, BamHI, or EcoRI techniques we have identified a significant proportion of (BRL), under conditions recommended by the manufacturer. Digested samples were separated by electrophoresis in 0.8% primary lymphoid neoplasms with derangements of the Rb agarose gels, according to standard technique^^^^^^ and transferred locus. As shown in studies of various carcinomas and sarcomas, nucleic acid hybridization methods detect only a subset of the tumors carrying mutations at the Rb locus. From the Laboratory of Pathology, National Cancer Institute, Studies of the Rb protein should show an even higher Na tional Institutes of Health, Bethesda, MD; and the Department of percent of lymphoid neoplasms associated with Rb mutaPathology, Georgetown Universiry, Washington, DC. tions. Thus, our findings support a role for inactivation of Submitted May 21, 1990; accepted October 22, 1990. the Rb gene product in the development of human lymAddress reprint requests to Jefiey Cossman, MD, Department of phoid malignancies. Pathology, 119 Basic Science Bldg, Georgetown University School of Medicine, 3900 Reservoir Rd, Nu! Washington, DC 20007. (Y

MATERIALS AND METHODS

Cells and tissues. Primary cases of malignant lymphoma and leukemia were collected and stored frozen as previously described.” Cell lines used in this study include the T-cell acute lymphoblastic leukemia (T-ALL) lines, HSB-2 (American Type Blood, Vol77, N o 4 (February 15). 1991: pp833-840

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section 1734 solely to indicate this fact. This is a USgovemment work. There are no restrictions on its use, 0006-4971/91/7704-0014$0.0010 833


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Fig 1. Analysis by Southern and Northern blotting of the Rb locus in a variety of lymphoid cell lines. (A) Genomic DNA map of the Rb IOCUS.’~~~ H, Hindlll restriction endonuclease cleavage sites. The normal sizes of the Hindlll-generated restriction fragments are indicated by the brackets below the map, in kilobases. Exons are indicated by black boxes (W) just above the map. Open rectangles above the map indicate the correspondence between the cDNA probes, Rb3.8 and Rb0.9, and the genomic DNA. (6) Southern blot. Genomic DNA from human placenta, a Burkin lymphoma cell line (Raji), five T-ALL cell lines (CEM, Jurkat, Molt-4, 8402. HSB-2) and human peripheral blood lymphocytes from t w o normal controls (PBL-1 and PEL-2) were digested with Hindlll and subjected t o Southern blot analysis with Rb3.8, as described in Materials and Methods. All control samples and cell lines except HSB-2 demonstrated the germline DNA configuration at the Rb locus. HSB-2 exhibits deletion of the lo-, 6.2-, and 2.1-kb bands, as well as one of the 5.4/5.3 and one of the 4.5/4.3 bands, corresponding t o exons 18 through 27 of the Rb gene. (C) Northern blot. Total cellular RNA, 15 pg, from five T-ALL cell lines (HSB-2, CEM, Jurkat, MoIt-4,8402) and from a B-cell line derived from the same patient as the HSB-2 cell line (SB) were electrophoresed and subjected t o Northern blot analysis, as described in Materials and Methods. The blot was first hybridized with Rb3.8, then washed and rehybridized t o a p-tubulin cDNA probe, as a control for equal loading of all gel lanes. Hybridization with Rb3.8 showed that HSB-2 does not synthesize detectable amounts of the normal 4.7-kb Rb transcript, but produces several aberrently sized Rb-hybridizing species, the most abundant of which is approximately 1.8 kb in total cellular RNA. SB cells, in contrast, synthesize abundant 4.7-kb Rb RNA. Jurkat cells do not synthesize detectable amounts of any Rb-hybridizing RNA species.


Re IN LYMPHOMA AND LEUKEMIA

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We next examined DNA from 26 primary cases of T-ALL to Genescreen Plus membranes, according to the manufacturer's by Southern hybridization analysis, using multiple restricdirections (New England Nuclear [NENIDuPont, Boston, MA). Hybridization to (a . 3ZP)dCTP-tabeledcDNA probes was pertion enzymes and probing with both Rb3.8 and Rb0.9. One formed according to the "4TC-formamide procedure" recomcase, no. 1709, demonstrated a germline configuration mended by NEN/DuPont, except that an additional highwhen probed with Rb0.9 (data not shown), but showed a 3' stringency wash in 0.1X saturate of sodium citrate (SSC)/O.l% deletion when probed with Rb3.8, identical at this level of sodium dodecyl sulfate (SDS) at 65°C for 20 minutes was generally analysis to the 3' Rb deletion present in HSB-2 cells. This added to the washing protocol. Southern blots were stripped of deletion is seen as a loss of the greater than 19- and 10.8-kb labeled probe by boiling for 20 to 30 minutes in 0.1X SSC/l.O% bands in BamHI-digested DNA, the 6.5, 4.1-, 3.0-, and SDS before rehybridization with a second probe. Filters were 2.3-kb bands in EcoRI-digested DNA, and the lo-, 6.2-, 4.3exposed for 16 hours to 8 days at -70°C with an intensifyingscreen. Northem blors. Total cellular RNA, 10 to 15 pg, was loaded per (partially lost, see above), and 2.1-kb bands in HindIIIlane on 1% formaldehyde-agarosegels and electrophoresed accorddigested genomic DNA (Fig 2A and data not shown). ing to standard conditions?'~" Postelectrophoresis, RNAs were Therefore, this primary case, like the HSB-2 cell line, has transferred to GeneScreenPlus according to manufacturer's direcapparently deleted exons 18 through 27 from both chromotions. Prehybridization conditions were as follows: 50% deionized somes. The remaining 25 cases all showed a germline DNA formamide, 1% SDS, 1 mom NaCI, and 10% dextran sulfate at configuration. Material was available to perform Northern 42°C for at least 2 hours. Probe (1 to 10 ng/mL) and denatured blot analysis on seven T-ALL cases, including case no. 1709, salmon sperm DNA (100 pg/mL) were added to the prehybridizaas well as two for whom DNA was not available for tion reactions and hybridizations performed for 16 to 24 hours at Southern analysis. No Rb-hybridizing RNA species were 42°C. Blots were washed twice in 2x SSC at room temperature, twice in 2x SSC/l% SDS at 60°C for 30 minutes, and once in 0 . 1 ~ detectable by Northern blot analysis in case no. 1709 (Fig SSC/O.l% SDS at 60°C for 10 to 20 minutes. Filters were exposed 2B). The six other cases all demonstrated significant and for 16 hours to 8 days at -70°C with an intensifying screen. approximately equivalent amounts of Rb 4.7-kb transcript (Fig 2B and data not shown). Thus, 1 of 26 primary cases of RESULTS T A L L showed an abnormal Rb locus. Chronic lymphocytic leukemia (CLL)lwell-differentiated We analyzed the Rb locus in a variety of lymphomas and lymphoma (WDL). We performed Southern blot hybridizaleukemias, examining both primary case material and cell tion analysis of genomic DNA extracted from 40 primary lines for deletions or mutations in the DNA and for cases of CLLWDL. One case, no. 1901, demonstrated a 5' alterations in Rb RNA expression. deletion at the Rb locus in both alleles, represented by loss 1 presents Southern blot and Northern Figure T-ALL. of the lo-, 8.7-, and 3.4-kb bands in EcoRI-digested blot hybridization data for five T-ALL cell lines: CEM, genomic DNA. The 2.8-kb band appeared to be present in Jurkat, Molt-4, 8402, and HSB-2. HSB-2 shows a homozyonly one copy (Fig 3A). The 39 other CLL cases showed a gous deletion with the 3' cDNA probe, Rb3.8, involving the germline DNA configuration at the Rb locus when probed lo-, 6.2-, 4.3- (partially lost only, due to comigration of with both Rb3.8 and Rb0.9. Three of these 39 cases, plus bands; see also reference 28), and 2.1-kb HindIII-generated two additional cases of CLL, were analyzed by Northern restriction fragments, which encode exons 18 through 27 blot hybridization. One of these five cases, no. 1483, (Fig 1A and B). This deletion has occurred despite retensynthesized markedly reduced levels of the Rb 4.7-kb tion of two cytogenetically normal chromosomes 13, a transcript, despite showing germline DNA configuration by finding independently confirmed by others.**The other four Southern analysis with both cDNA probes, using EcoRI-, cell lines show the germline configuration of the Rb locus HindIII-, and BamHI-digested genomic DNA (Fig 3 and when probed with both 5' and 3' probes encoding the entire data not shown). In the other four cases significant amounts cDNA sequence4 (Fig 1). Northern blot analysis of total of an apparently normal 4.7-kb Rb transcript were detected cellular RNA from these cell lines (Fig 1C) showed abun(Fig 3B). These five cases were all similarly estimated to dant wild-type sized, 4.7-kb transcripts in CEM, Molt-4, contain at least 70% to 80% tumor cells by immunophenoand 8402 cells. In HSB-2, several hybridizing species are typic and flow cytometric analysis. seen: a minor band at approximately 6 kb representing an apparently unpolyadenylated form,= and several smaller Intermediately differentiated lymphoma (IDL). We analyzed 15 primary cases of IDL, a low-grade B-cell neospecies, the most abundant of which is approximately 1.8 kb. SB, a B-lymphoblastoid cell line derived from the same p l a ~ m , ~by ~,~ Southern ' blot hybridization. One case, no. patient as the HSB-2 cell line, shows a germline DNA 1869, demonstrated a deletion in the Rb locus. As seen in Fig 4A, a Southern blot of HindIII-digested no. 1869 DNA configuration at the Rb locus and abundant 4.7-kb Rb RNA (Figs 1C and 2A, and data not shown). Therefore, the probed with Rb3.8 revealed that all Rb3.8-hybridizing deletion in the Rb locus in HSB-2 cells appears to be tumor bands showed decreased signal intensity, consistent with loss of exons 10 through 27 from at least one chromosome specific. In Jurkat cells, which do not have a detectable in the cells of this patient's neoplasm. Because this is deletion by Southern blot analysis (Fig IB), little or no Rb message was observed in Northern blots with either cDNA primary patient material, one cannot be sure whether the relatively faint signals present at germline band sizes probe, Rb3.8, or Rb0.9 (Fig 1 and data not shown). Two of five T-ALL cell lines examined (HSB-2 and Jurkat), thererepresent Rb DNA from one undeleted chromosome in the fore, show markedly decreased to no detectable wild-type malignant cells or DNA contributed by admixed benign Rb expression at the RNA level. cells. Flow cytometric analysis of both this case and case no.


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Actin Fig 2. Southem and Northem blot analysis of the Rb locus in T-ALVLBL. (A) Southem blot. Genomic DNA, 10 pg, from a B-cell line (SB), a T-ALL cell line (HSB-2). and a prhnary case of T-ALL (no. 1709) were digested with BamHl (left panel) and Hindlll (right panel), and subjected t o Southern blot analysis with the cDNA probe Rb3.8 (see Materials and Methods). SB DNA exhibits the germline DNA configuration. HSB-2 and no. 1709 DNAs show loss of all but the approximately 7.8-kb band in BamHI-digested DNA, and of all but the 7.5- and one of the 5.3-kb bands (indicated by arrows) in Hindlll-digested DNA (longer exposures show one of the 4.544.3-kb bands as well; see also Fig lB, HSB-2 lane). DNA sizes (in kilobases) are indicated (left panel, molecular weight markers are BRL "1 kb ladder"; right panel, germline DNA bands). (B) Northern blot. Total cellular RNA, 15 pg, from t w o primary cases of T-ALL (nos. 1709 and 864) and one case of WDL (for comparison; no. 2010) were subjected t o Northern blot analysis, as described in Materials and Methods. The filters were first hybridized with Rb3.8, then stripped and rehybridized with a chicken actin probe, as a control for equal lane loading. The T-ALL case no. 1709 exhibits little or no detectable 4.7-kb Rb RNA, even on approximately fivefold longer film exposures.

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- 10 -8.7 -7.5 Fig 3. Southern and Northern blot analysis of the Rb locus in CLUWDL. (A) Southern blot. Genomic DNA, 10 pg, from primary cases of CLL were digested with EcoRl and subjected t o Southern blot analysis with the 5' cDNA probe Rb0.9, as described in Materials and Methods. Case no. 1901 demonstrates loss of the lo-, 8.7-. and 3.4-kb EcoRl bands. The 2.8-kb band appears t o be present at half intensity, suggesting deletion from only one chromosome. All other cases of CLL examined displayed germline configuration with both Rb cDNA probes. Sizes of germline DNA bands are indicated (in kilobases). (6) Northern blot. Total cellular RNA, 15 bg, extracted from five primary cases of CLL were electrophoresed and subjected t o Northern blot analysis, first with Rb3.8 and then with a p-tubulin cDNA probe, as a control for equal loading of lanes (see Materials and Methods). Case no, 1483 shows markedly reduced levels of Rb 4.7-kb RNA relative to the other cases. Case no. 752 is relatively underloaded.

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2086 (see Fig 4A) demonstrated that the malignant cells represent 70% to 80% of the total cell population in each of these tumors. Rehybridization with a control probe from chromosome 18 (see lower panel of Fig 4A) showed equivalent loading and integrity of DNA samples on this Southern blot. Hybridization with Rb0.9 demonstrated 5' Rb DNA sequences in a germline configuration. Assessment of relative signal intensity of the 5' bands is rendered difficult by cross-hybridization of Rb0.9 probe sequences to repetitive sequences in the genomic DNA.4 Therefore, we cannot conclude with certainty whether one chromosomal copy of these 5' sequence bands is deleted as well. However, Northern blot analysis of no. 1869 RNA showed undetectable levels of any Rb-hybridizing transcript, suggesting that both copies of the Rb gene are defective in this

patient's malignant cells (Fig 3B). Five other cases of IDL examined by Northern blot analysis demonstrated easily detectable amounts of an Rb-hybridizing 4.7-kb transcript. B-cell lines. The B-lymphoblastoid cell lines SB and 8392, and the Burkitt lymphoma cell lines, Raji and Daudi (Figs 1 and 2, and unpublished data) were also examined and shown to contain Rb DNA in the germline configuration and significant amounts of 4.7-kb Rb transcript. DISCUSSION

We have examined a total of nine lymphoid cell lines and 85 primary cases of lymphoid malignancies for alterations in Rb DNA and/or Rb RNA expression. Two of the cell lines and four of the primary cases were abnormal in these

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only. GINSBERG, RAFFELD, AND COSSMAN

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(bcl-2) assays. In view of the relatively insensitive nature of these nucleic acid hybridization techniques, these results show a significant mutation frequency in the Rb locus in both lowand high-grade lymphoid neoplasms. Nucleic acid hybridization studies of a variety of human solid tumors have

Fig 4. Southern and Northern blot analysis of the Rb locus in primary cases of IDL. (A) Southern blot. Genomic DNA, 15 pg, from primary cases of IDL were digested with Hindlll and subjected t o Southern blot analysis, as described in Materials and Methods. The blot was probed sequentially with a chromosome 18, single-copy gene probeM(belZ ) , Rb3.8, and Rb0.9. Hybridization with the chromosome 18 probe shows approximately equivalent loading of all gel lanes. Hybridization with the Rb3.8 probe showed relatively decreased signal intensities of all 3' bands in DNA from case no. 1869, indicating deletion of the Rb gene from one or both chromosomes, in at least a subset of the cells in this patient's material (see Discussion). (B) Northern blot. Total cellular RNA, 15 pg, from two primary cases of IDL (nos. 1919 and 1869) and one case of WDL, for comparison (no. 2010). were subjected t o Northern blot analysis, sequentially with chicken actin and Rb3.8 cDNA probes, as described in Materials and Methods. Probing with the actin probe showed approximately equal loading of all gel lanes. Probing with Rb3.8 demonstrated that case no. 1869 synthesizes markedly reduced amounts of Rbhybridizing RNA.

demonstrated alterations in Rb DNA or RNA in only a small percent of primary cases (eg, studies of lung, breast, and bladder cancers), whereas subsequent studies of Rb protein in these tumors have frequently shown a much higher frequency of Rb abnormalities." Therefore, it is of

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Rs IN LYMPHOMA AND LEUKEMIA

particular interest now to examine the status of the Rb protein in lymphoid neoplasms. It has been proposed’*”’”*that inactivation of the Rb locus may contribute to the progression rather than initiation of many solid tumors. Thus, it is noteworthy that of three primary cases of low-grade lymphomas with abnormalities in Rb, two exhibited histologic features considered to be poor prognostic indicator^.'^.^^.'^ Patient no. 1869 exhibited IDL in blast crisis and patient no. 1483 exhibited WDL with an unusually high mitotic rate. Clinically, both of these patients did exceptionally poorly* (the third patient, no. 1901, died shortly after his diagnosis of CLL, but of causes unrelated to this disease). Furthermore, T-ALL, a highgrade lymphoid neoplasm, demonstrated Rb alterations by Northern blot analysis in one of seven primary cases and two of five cell lines. Therefore, our findings suggest that mutational inactivation of the Rb locus contributes to the progression of lymphoid neoplasms from low-grade to aggressive subtypes. Studies examining primary case material for the absence of DNA or RNA sequences are complicated both by variable amounts of nucleic acid degradation and by the *Patient no. 1869 was diagnosed with stage IVA IDL, having a positive bone marrow, but negative liver and spleen at presentation, yet died within 21 months of diagnosis despite aggressive combination chemotherapy. The reported median survival for stage IVA IDL is greater than 5 years.” Patient no. 1483 was diagnosed with Rai stage I CLL at the age of 55 years; he died only 30 months later because of complications of his disease. Median survival for patients with stage I CLL is approximately 51 months?l” The other patient with CLL/WDL demonstrating mutated Rb locus, no. 1901, died 11 months after diagnosis, but of causes unrelated to his lymphoma.

presence in these specimens of admixed normal cells. We attempted to minimize these problems by excluding any samples that demonstrated significant amounts of degradation and by examining lymphomas and leukemias that characteristically contain relatively few admixed benign cells. The use of in situ techniques will lessen the endemic problem of signal contribution from contaminating benign cells by helping to delineate whether small but detectable amounts of Rb RNA seen in patient materials (eg, see case no. 1483) represent contaminating RNA from admixed benign cells, Rb expression in only a subset of malignant cells, or markedly decreased amounts of normal gene transcript being synthesized by all malignant cells. We have shown mutations at the Rb locus by Southern analysis in 1 of 5 high-grade IymphomaAeukemia (T-ALL) cell lines, in 1of 26 primary cases of high-grade lymphoma/ leukemia, and in 1 of 55 primary cases of low-grade B-cell malignancies (CLL/WDL and IDL). By Northern analysis, we detected markedly decreased or no Rb transcripts in 2 of 5 high-grade leukemia cell lines, 1 of 7 primary cases of high-grade lymphomaAeukemia, and 2 of 11 low-grade B-cell malignancies. Future studies using in situ and antibody techniques should show a role for abnormal Rb expression in more cases of lymphoid malignancies and help to test the model that inactivation of Rb contributes to progression in human lymphoma and leukemia.

ACKNOWLEDGMENT

We thank Dr S. Friend for generously providing the Rb cDNA probes; Dr C. Croce for the bcl-2 probe; Dr Andrew Larner for the actin and tubulin probes; and Drs Han van Krieken and Jeffrey Madeiros for contributing two of the Southern blots used in this study.

REFERENCES

1. Knudsen AG Jr: Mutation and cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci USA 68:820,1971 2. Godbout R Dryja TP, Squire J, Gallie BL, Phillips R A Somatic inactivation of genes on chromosome 13 is a common event in both hereditary and nonhereditary retinoblastoma tumors. Nature 304:451,1983 3. Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL,Strong L, White RL: Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305:779,1983 4. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja TP: A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323643, 1986 5. Lee WH, Bookstein R, Hong F, Young LH, Shew JY, Lee EY-HP: Human retinoblastoma susceptibility gene: Cloning, identification and sequence. Science 235:1394,1987 6. Ludlow JW, Shon J, Pipas JM, Livingston DM, DeCaprio J A The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T. Cell 60:387,1990 7. Buchkovich K, Du@ LA, Harlow E: The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Celi 58:1097,1989

8. Chen P-L, Scully P, Shew J-Y, Wang JYJ, Lee W-H: Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell 58:1193,1989 9. Mihara K, Cao XR, Yen A, Chandler S, Driscoll B, Murphree AL, T’Ang A, Fung Y-KT: Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science 246:1300,1989 10. Fung YKT, Murphree AL, T’Ang A, Qian J, Hinrichs SH, Benedict W F Structural evidence for the authenticity of the human retinoblastoma gene. Science 236:1657,1987 11. Weischselbaum R, Beckett M, Diamond A Some retinoblastomas, osteosarcomas, and soft tissue sarcomas may share a common etiology. Proc Natl Acad Sci USA 85:2106,1988 12. Reissman PT, Simon MA, Lee W-H, Slamon DJ: Studies of the retinoblastoma gene in human sarcomas. Oncogene 45339,1989 13. Toguchida J, Ishizaki K, Sasaki MS, Ikenaga M, Sugimoto M, Kotoura Y, Yamamuro T Chromosomal reorganization for the expression of recessive mutation of retinoblastoma susceptibility gene in the development of osteosarcoma. Cancer Res 48:3939, 1988 14. Xu H-J, Hu S-X, Hashimoto T, Takahashi R, Benedict WF: The retinoblastoma susceptibility gene product: A characteristic pattern in normal cells and abnormal expression in malignant cells. Oncogene 4:807,1989

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