Mechanisms in B-cell neoplasia

ImmunologyToday,voL 7, No. 9, 1986

Fundamental changes occur in cells of the B lymphocytic lineage which lead to uncontrolled cellular proliferation. Mechanisms that deregulate the c-myc oncogene are particularly important in neoplastic development. These and other influences on B-cell tumor growth were discussed at a recent meeting*. In 1981 Hayward, Neel and Astrin discovered retroviral promoter insertions in bursal lymphomas that affected c-myc transcription. This was soon followed by the realization that specific chromosomal translocations rearranged and deregulated cmyc in the mouse, human and rat B-lineage tumors (Table 1). Universal agreement on the identity of the critical biological consequences of c-myc deregulation is not yet reached. It seems generally accepted that c-myc is transcribed in many proliferating cells. In neoplastic B cells, the level of transcription of c-myc is usually higher (though variable) than in normal proliferating B cells. There was growing acceptance of the notion that c-myc expression is deactivated when a cell enters a Go-like state. Deregulation of c-myc expression, therefore, prevents the normal deactivation process and is dominant over the normal regulation of c-rnyc expression. As one possible consequence, neoplastic B cells cannot exit from continuous cycling, At least three times during the development of normal B cells from stem cells a resting state is reached where proliferation ceases. The first resting state occurs when pre-B cells become surface IgM (and often IgD)positive, antigen-sensitive B cells. It appears that transgenic mice carrying E~-c-myc in the germline accumulate cycling pre-B cells in the pre-neoplastic period of their development (A. Harris and J. Adams, Melbourne). The transition from a cycling pre-B cell to a resting B cell may, therefore, be the first stage in normal B-cell development at which deactivation of c-myc expression leads to a resting state. Once B cells have become resting, they must be activated by either antigen, with or without the help of T cells, or by polyclonal activators such as Igspecific antibodies, bacterial lipo-

*The4th Workshopon Mechanismsin B-cellNeoplasia was held in Bethesda,Maryland, 24-26 March,1986.

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Mechanisms in B-cell neoplasia from Kenneth Marcu, Fritz Melchers, Herbert C. Morse, III and Michael Potter polysaccharides (LPS) or bacterial lipoproteins, to enter the mitotic cycle again. During this second proliferative phase, B cells can switch expression of the class of heavy chains and can hypermutate Ig variable-gene segments. Two types of resting stages emerge. One is the surface-lg-positive memory cell, often expressing classes of somatically mutated Ig other than IgM or IgD. The other is the Ig-secreting plasma cell, which is generated by maturation events which increase in influence with successive divisions and which appear antagonistic to the capacity of a B cell to proliferate. More and more endoplasmic reticulure is formed, on which more and more secretory-type Ig mRNA is translated. Plasma cell tumors producing and secreting IgM exist which appear 'frozen' at different stages of maturity of this development. A major question is when c-myc deregulation occurs in the natural history of B-cell tumor formation.

Evidence accumulates that deregulation of c-myc may not itself transform B lymphocytes into an autonomously proliferating cell type but may cause instead an abnormal expansion of a population of cells. Avian bursal lymphomatosis has defined morphological stages (transformed follicles ~ bursal nodules --> lymphoma). Only a few transformed follicles progress to bursal nodules (E. Humphries, Dallas). When chick embryos are treated with cyclophosphamide and infected with wild type MC29 virus, no follicles form; however, when infected with a variant MC29 virus HB-1 which has a hybrid v-mydc-myc gene, large numbers of transformed follicles form, but not bursal nodules (P. Nieman, Seattle). Sequence analysis demonstrated that HB-1 had a myc gene made up of viral and cellular sequences and when injected into young birds gave T and B cell lymphomas and an occasional plasmacytoma, in contrast with wild type

Table 1. B-celltumor modelsystems

B-celltumor

mycgenederegulation

Mechanisms possibly implicated in pathogenesis

Burkitt's lymphoma(BL)

t(8;2), t(8;22), t(8; 14) rcpt translocations(100% of cases) t(8;14) translocation(60% of cases) Noneyet consistently demonstrated Noneyet consistently demonstrated Insertionof ALV promoters

Chronicmalaria(endemic) EBVinfection

Follicular lymphoma(FL) Multiple myeloma(MM) B-chroniclymphocytic leukemia(B-CLL) Chickenbursallymphomas (CBL) Rat immunocytomas(R-PCT)

t(6; 7) translocations(I 00% of cases)

0il-induced mouse plasmacytomas(M-PCT)

rcpt (12; 15) rcpt (6; 15) et al. (95% of cases)

Pristane-J-3induced plasmacytomas(J-3PCT) El~-c-myctransgenicmice

avianv-myctransduction

~ 1986, Elsevier Science Publishers B.V., Amsterdam 0167 - 4919/86/$02.00

El~-c-mycgenomic insertion

Unknown Unknown Mutations inducing block differentiation ALV infection Possiblyparasiticinfections (40% of tumors produce IgEmyelomaproteins) Radicalproductionfrom oil phagocytosis; possible geneticdefectin DNA repair Unknown Greatlyexpandedpre-B population 249

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MC29 which gave tumors of myeloid origin. This suggests that sequence changes in the myc gene can change the target cell specificity of the virus (P. Enrietto, Stony Brook). In mouse ptasmacytomagenesis, there are proliferations Of plasma cells (proliferative foci) that can be found long before the plasmacytomas develop. These cells have not yet been shown to have translocations but evidence from Abelson virus pristane induction experiments suggests the targets of Abelson virus introduced at day 40 may be cells which already have chromosome 15 translocations and associated myc gene deregulations (M. Potter et al., Bethesda). In contrast, infection of mice a week or so after pristane with J-3 virus (a retroviral construct containing avian v-myc) induces plasmacytomas with intermediate latent periods (mean of 73 days; M. Potter and H.C. Morse, Bethesda). Interestingly, most of these pristane/J-3 virus induced tumors lack chromosome 15 translocations. Since infection by the J-3 virus puts an avian v-myc gene under the control of viral long terminal repeat (LTR) promoters, the intermediate latent period suggests that autonomous transformation has not yet occurred and that additional changes for transformation to autonomy are required. Curiously, other retroviral constructs containing c-myc (W. Baumbach, Princeton; L. Wolff, Bethesda) have produced only myeloid tumors. This suggests that J-3 may have special biological properties. Possibly these are related to the partial origin of v-myc in J-3 from MH2 virus or the presence in this virus of partial raf sequences (U. Rapp, Frederick). Many of the transgentc mice carrying Et~--c-myc sequences introduced into the germline do not develop monoclonal tumors until 6 to 8 weeks of age or longer (J. Adams and A. Harris). Since these transgenic mice have hyperplastic expansions of the pre-Bcell population in the bone marrow and spleen during this pre-neoplastic period, deregulated c-myc genes may not transform B lymphocytes in a single step and critical additional changes may be required. These remain to be identified. The general picture that emerges from the model system is that c-myc deregulation probably occurs as an early permissive event in the progression towards neoplasia but that by itself it does not transform B lympho-

ImmunologyToday,vol. 7, No. 9, 1986

cytes to autonomy. A similar process pancy of surface Ig. It remains to be may occur in mice infected with seen whether the deregulation of murine retroviruses containing avian BSF-1 expression or its receptors v-myc (H. Morse) or MTV/myc trans- could contribute to B-cell neoplasia. genic mice (P. Pattengale, Boston) Progression of activated B cells that were shown to develop a wide through successive cell cycles is conrange of hematopoietic tumors (T trolled in a synergistic fashion by cell, B cell, pre-B cell), mastocytomas three interactions with ligands of and solid tumors (mammary and three origins at three points in the pancreatic adenocarcinomas, Sertoli cell cycle (F. Melchers, Basel). Anticell tumors). gen binding to Ig and helper T cell binding to antigen plus MHC II molePhysiologyof B-cellactivation cules on B cells control entry in G1 The search for additional deregu- directly after mitosis. This is compalations on the way to full malignancy rable with the action of PDGF as a could be aided by knowledge of the competence factor in growth control processes which control activation of of fibroblasts where intracellular signormal resting B cells and cell cycle nalling appears to be mediated by an progression of their activated coun- initial activation of phospholipase C terparts. Prolonged stimulatory in- (J. Campisi). The second restriction fluences on B cells by viruses such as point occurs near the boundary of Epstein-Barr virus (EBV) and parasites G1 to S transition and is controlled by such as malaria are likely to increase a set of factors Melchers calls the chances for additional deregu- factors. Cross-linked forms of C3b lations. Deregulations in the B-cell and C3d have c~ factor-replacing growth-supporting environment will activity for B cells, which implicates also provide chronic B-cell stimulat- the C3d-specific CR2 receptor as a ing conditions. Motheaten mice signal receiver. This has prompted a have unusually high levels of B-cell search for C3 synthesis in c~ factormaturation factors and 50-fold devi- producing cell lines. Two C3 specificated levels of IgM in the serum. They mRNA species (1.8 kilobases and 4.2 may represent a case of an inherited kb) are detected with C3-specific deregulation of the stimulatory en- DNA probes in all ~ factor-producing vironment for B cells (C. Sidman, Bar lines (macrophages, T and B tumors) Harbor). but not in ~ factor-negative cells Antigen-induced B-cell growth be- (certain lines, normal B cells) (W. gins with the uptake, processing and Lernhardt, La Jolla). The normal size presentation of antigen by acces- of C3-specific mRNA found in liver is sory, antigen-presenting (A) cells. 5.6 kb. It remains to be established The processed antigen is presented whether these mRNA molecules in the context of class II MHC mole- code for protein, whether such procules to antigen-specific, MHC- teins have ~ factor-like activities, and restricted helper T cells. The T celI-A how they relate to the known forms cell interaction leads to activation of of C3. CR2 is also the receptor for EBV on both types of cells, and the production of lymphokines, each cell type human B cells. EBV may, therefore, producing a different set. Resting B overcome the requirement for c~ faccells are, however, refractory to tors in the initial phase of growth of the action of the proliferation- the transformed B cells, although the promoting lymphokines. B cells have problem of EBV transformation is to be excited by the binding of anti- certainly much more complex, since gen to Ig, or by factors produced by only resting, but not activated B cells helper T cells responding to antigen are infected by the virus (P. Aman, plus MHC class II molecules, to be Stockholm), and since latentlystimulated through the cell cycle. infected B cells can also grow withBSF-1, a 20 kDa glycoprotein which out the help of ~ factors (E. Klein, has recently been sequenced (R. Stockholm). Many plasmacytomas which are Coffman, Palo Alto) appears to control an early step in the Go --~ G1 propagated in vivo by transfer in transition. However, it does not acti- inbred mice can be grown in vitro, vate protein kinase C (W. Paul, but only when ~ factor-like activities, Bethesda) as platelet-derived growth produced by human endothelial cell factor (PDGF) would do in fibroblasts cultures (L. Aarden, Amsterdam) or (J. Campisi, Boston). It is, therefore, the macrophage line P388D1 (R. acting presumably at an earlier stage Nordan, Bethesda), are present. The of B-cell activation, before the occu- nature of these factors, 21 and 24

ImmunologyToday,voL 7, NO.9, 1986

kDa in size, is still unknown. IL-1, IL-2, IL-3, BSF-1, epidermal growth factor (EGF), tumor cell growth factors, colony stimulating factor, B-cell growth factors (BCGF) I and II, interferon--y, C3b, thymosin, concanavalin A and lipopolysaccharide have been excluded. The action of these factors on plasmacytoma cells is in the G1 phase of the cell cycle, It remains to be seen how these tumor growth factors relate to the ~ factors acting on normal B cells. A third restriction point in the cell cycle is observed late in G2, before entry into mitosis. It appears to be controlled by factors Melchers calls [3 factors, which are produced by helper T cells. A variety of candidates exists at present for such activities: BCGF-II, IL-2 and, recently, eosinophil growth factor (A. O'Garra, London). One important question which remains unanswered is whether deregulation from a ligand-requiring to a ligand-independent state of signalling at any one of the three restriction points in the B-cell cycle leads to premalignant states of B cells. (NZB x NZW)F1 and BXSB mice, which show B-cell hyperplasias and which appear deregulated at one or two, but not all three restriction points (A. Rolink and F. Melchers, Basel) may be interesting cases in the search for inherited pre-neoplastic states of B cells. It will certainly be worthwhile to study the effect of introducing a constitutively-expressed c-myc gene into hematopoietic stem cells, or B cells of such mice, on the kinetics of development and the characteristics of B-cell neoplasms. Mechanisms of chromosomal translocation The biological changes which lead to translocation are not known. The prevailing notion is that chromosome breaks and rejoinings occur continuously and stochastically. The possibility that certain types of rearrangements are differentiationdependent was discussed by I. Kirsch, Bethesda. This could, of course, explain the involvement of Ig loci in B-cell tumors. Mechanisms that repair double-stranded chromosome breaks may influence rejoinings. Double-stranded breaks induced by X-irradiation or light during G2 are normally repaired rapidly in plasmacytoma-resistant strains such as BALB/cJ, DBA/2 and (BALB/cAn x DBA/2)F1 while a de-

news aM[eatures 7 layed repair was demonstrated in the plasmacytoma susceptible BALB/cAn strain (K. Sanford, Bethesda; R. Parshad, Washington, DC). Failure of normal repair processes may increase the possibilities for recombination associated with the activity of B-cell specific recombinases.

In-vitro targets for transforming viruses Earlier studies of adult bone marrow or fetal liver cultures and more recently long-term Whitlock-Witte bone marrow cultures infected with murine leukemia viruses (MuLV) containing abl or fes showed that only pre-B cell tumors were recovered readily. Data presented at this workshop demonstrated that pre-B cell lymphomas were also induced from bone marrow or fetal liver cultures by a murine retrovirus construct containing the avian v-erbB oncogene (J. Pierce, Bethesda). Soft agar colonies of cells infe'cted with this construct differed from ab/-induced colonies in that they could be expanded without the use of feeder layers. Newborn mice infected with this virus also developed pre-B cell lymphomas with short latencies. Pre-B cell lines with autonomous growth potential in vitro and in vivo were obtained from Whitlock-Witte cultures infected simultaneously with one MuLV containing H-ras and a second containing an avian v-myc gene (O. Witte, Los Angeles). Cells from cultures exposed to ras alone demonstrated sustained growth in vitro but not in vivo whereas there were no effects on cultures exposed to myc alone. This suggestion that synergistic interactions of more than one onc gene may be required to elicit autonomous growth was supported by the observations that bone marrow or fetal liver cells infected with a single virus containing both raf and an avian v-myc yielded continuous macrophage cell lines and that one of these lines had a rearranged myb gene resulting from reintegration of a helper virus (J. Cleveland, Frederick). Additional indications of a requirement for the activation of two onc genes in neoplasia derive from studies of DNA from ablinduced tumors that induced transformation of NIH-3T3 cells in spite of the fact that v-abl could not be detected (R. Risser, Madison). The data in these reports can be summarized to suggest that while an unexpectedly wide variety of onc genes can initiate the transformation

of the B-cell and myelomonocytic lineages in vitro, additional alteration of the genome, most likely affecting the expression of other one genes, is required for outgrowth of clonal neoplastic cells. Proliferation and c-mycexpression in normal and transformed cells Several presentations dealt with the effects of receptor-transduced environmental stimuli on the expression of proto-oncogenes, with particular emphasis on c-myc. Expression of c-myc and c-fos in cultured A31 BALB/c fibroblasts can be induced by independent pathways which may reflect different states of cell physiology (J. Campisi). Serum (i.e. PDGF) and tetraadecanoylphorbol acetate (TPA) are efficient inducers of c-myc and c-fos in densityarrested, confluent cells while EGF is ineffective. However, serumdeprived, subconfluent cells are equally responsive to serum and EGF while TPA is a poor inducer with regard to myc and los activation. Serum and TPA signals are transduced through the C kinase pathway. The EGF effect does not involve C kinase but could result in increases in extracellular calcium and may be manifested in cooperation with cyclic AMP. Transcription of c-myc is activated in normal human B lymphocytes by anti-lg and monoclonal antibodies directed against different cell surface antigens while only the anti-lg signal synergizes with B-cell growth factor to allow for cell cycle progression (E. Smeland, Oslo). Anti-lg and TPA were also shown to induce c-myc transcription in murine WEHI 231 B lymphoma cells (G. Sonnenshein, Boston) and in the BJAB human lymphoblastoid line (Aman) which both cease proliferating around 18 h after anti-lg treatments. The anti Iginduced transient accumulation of c-myc RNA in WEHI 231 reflects alterations in both transcriptional and post-transcriptional controls of cmyc expression. In situ hybridizations performed with rapidly proliferating tissues of the human embryo revealed that cytotrophoblasts express high levels of c-myc and c-sis (PDGF) while syncytiotrophoblasts do not. Expression of c-myc is therefore not characteristic of all types of proliferating cells in vivo (R. Ohlsson, Umea). Fetal calf serum induces c-myc expression in syncytiotrophoblasts in vitro while

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after 6 h of incubation less than 1% acting putative regulatory elements of cells in S phase express c-myc within the mouse and human c-myc protein. One interpretation of this genes were discussed. A DNA segfinding is that c-myc expression may ment located 400-1200 bp 5' of be Gl-restricted in vivo, as suggested mouse c-myc has the properties of a by earlier in vitro experiments on transcriptional 'dehancer' inhibiting density-arrested murine fibroblasts. SV40 enhancer-driven expression, However, the inability of tissue- independent of its orientation and derived syncytiotrophoblasts to proli- distance from a heterologous proferate in vitro may also be moter measured by $1 nuclease responsible for this observation. mapping (K. Marcu, Stony Brook). C-myc expression is also largely The myc 'dehancer' also reduced the restricted to the G1 phase of the activity of a myc-CAT (chloramphencell cycle in developing bursal icol acetyl-transferase) hybrid gene lymphocytes (C. Thompson, Seattle). transfected into BJAB human lymphoInterestingly, transformed bursal blastoid cells. DNA competition excells harboring a retroviral insertion periments performed with a human near c-myc display a high level of myc-CAT expression vector and myc expression late in S phase, various segments of the c-myc gene suggesting that inappropriate as competitors in a human Burkitt expression of this oncogene in vivo lymphoma line localized putative cismay somehow contribute to the acting negative regions with c-myc transformed phenotype (Thompson). exon 1 and intron 1 which presumably Inducers of differentiation of MEL normally interact with transacting (murine erythroleukemia) cells dram- negative regulatory factors (J. Chung, atically reduce c-myc expression at Boston). However, analogous comthe post-transcriptional level. One petition experiments performed with MEL cell line expressing a high level 5' flanking myc sequences revealed of a transfected human myc gene no other negative elements with (30-fold higher than endogenous c- similar properties. In contrast with myc) continued to proliferate in re- these results, an independent study sponse to differentiation inducers, of human myc-CAT genes containeven though the human myc trans- ing different lengths of 5' flanking cripts were partially down-regulated DNA revealed a putative c/s-acting (S. Segal, Bethesda). negative element 100-400 bp 5' of Consistent, non-random trans- the first c-myc promoter (R. Dallalocations of chromosome 8 and c- Favera, New York). A clear picture of myc deregulation are not a feature the physiological role of each of of chronic lymphocytic leukemia these putative negative elements is (CLL) and the chronic phase of multi- yet to emerge from these transfecple myeloma (MM). Rare variants in tion experiments. It should be consiMM (i.e., processes associated with dered in this context that nuclear effusions, local tumor formation or run-on experiments have revealed a plasma cell leukemia) may have dis- complex set of multiple, bidirectional orders of c-myc. G. Hollis (Bethesda) transcripts with the murine c-myc described an MM cell line in which a locus which appear to be dif3' rearrangement of c-myc was ferentially regulated (Marcu). It found. The treatment of the patient would appear that c-myc possesses with potentially mutagenic chemo- a variety of independent c/s-acting therapeutic agents may have contricontrol elements and we can expect buted to the development of this their characterization to intensify at aggressive cell variant. future workshops. K. Nilsson (Uppsala) has used the 1-73 B-CLL cell line to characterize Evidencefor autoregulation of c-myc mycfunction. Unstimulated 1-73 cells expression are arrested in Go but can be inThe first hint that c-myc may be duced to differentiate to secrete IgM autoregulated arose when the norby TPA. Concomitantly, they trans- mal alleles of plasma cell tumors cribe c-myc during GI. Thus, myc were found to be silent. Similar expression does not completely in- findings were subsequently reported hibit the development of a differen- for the untranslated myc alleles in tiated function. Burkitt lymphomas (BL). A competing hypothesis was formulated, Regulatory elementswithin the c-myc which suggested that c-myc is not locus expressed in the normal differenEvidence for a complex set of c/s- tiated (presumably non-dividing)

ImmunologyToday,voL 7, No. 9, 1986

counterparts of these transformed B cells, implying that the translocation event turns on a silent protooncogene. A number of speakers at this workshop provided additional evidence for the autoregulation model. Myeloid tumors obtained by infecting mice with murine retroviruses, expressing high levels of v-myc or c-myc genes, did not accumulate endogenous c-myc RNAs (Cleveland; Baumbach). Similar results were reported for lymphoid tumors derived with v-myc containing retroviruses (Morse). Immature and mature B-cell tumors derived from transgenic mice harboring a c-myc gene driven by the immunoglobulin heavy chain enhancer (El~-myc mice) did not express normal c-rnyc transcripts. However, autoregulation was not evident in two other instances. Endogenous c-myc expression was unaffected in a MEL cell line expressing a 30-fold excess of a transfected human myc gene (Segal). In addition, FR3T3 rat fibroblasts expressing a 50-100-fold excess of a murine c-myc gene in a retrovirus vector also maintained expression of their c-myc genes, even though the extremely high level of exogenous myc contributed to their transformed morphology, growth in agar and tumorigenicity (Marcu). The cellular context may therefore also contribute to this phenomenon, implying that downregulation of c-myc is not mediated directly by abnormally high levels of the c-myc gene product.

Mechanismsof oncogene activation in lymphoid neoplasia Previous workshops were largely concerned with mechanisms of oncogene activation in murine plasma cell tumors (PCT) and human BL and a number of presentations in this fourth meeting were also devoted to these topics. The molecular mechanism(s) underlying the involvement of the immunoglobulin heavy chain locus (Igh) in myc activation have not been established, since the known Ig enhancer element is generally not found on the same chromosome as the translocated myc genes. However, recent findings on Ig gene expression suggest that this enhancer is not required for the maintenance of the transcriptional competence of the Ig locus in B cells. Evidence was presented for the existence of promoter elements in the vicinity of the murine C3,2a gene that contributed to the expression of a

ImmunologyToday,voL 7, NO. 9, 1986

news translocated myc gene in a PCT. One Munich). Breakages in the vicinity of of these aberrant IgCH promoters JH heptamer-nonamer sequences may be analogous to the octamer were presented for 14;18 translocasequence (AITrGCAT) commonly tions in human follicular cell lymphofound in the B-cell specific promoter mas that occur near the bcl-2 locus of VH genes (Marcu). Additional evi- on chromosome 18 (Y. Tsujimoto, dence supporting the idea that re- Philadelphia; S. Korsmeyer, Bethescombination with the IgCH lOCUSdis- da). The bcl-2 locus contains at least rupts normal c-myc control comes two hot spots for 14;18 translocafrom the discovery of a clustered set tions and produces two overlapping of translocations within 1.2 kb 5' of transcripts which differ at their 3' c-myc in rat immunocytomas (W. ends due to differential splicing Pear, Stockholm). Secondary muta- (Tsujimoto). Bci-2 transcripts are extions localized at the exon 11intron I pressed in human pre-B and folliboundary in the translocated myc cular cell lymphomas but not in huloci of two PCT were found to pre- man myeloma or EBV-transformed vent normal c-myc splicing, thereby lymphoblastoid lines, suggesting resulting in the accumulation of very that its expression may normally be high levels of abnormally large cyto- restricted to early stages of B cell plasmic myc RNA (S. Bauer, Bethes- development (Korsmeyer). A novel da). These secondary mutations may 8;14 translocation was also denot only contribute to higher RNA scribed which involved the 3' flank stability but also to an increased of the c-myc gene and the conserved transcription rate through the c-myc recombination sequences of the Tlocus. The presence of an intact first cell receptor C~ locus (J. Erikson, exon in one of these large myc RNAs Philadelphia), providing additional rules out the possibility that this exon evidence for the role of lymphoid is the primary determinant of normal cell-specific recombinases in the c-myc RNA instability. This conclu- genesis of these translocations, sion was also supported by studies on transcripts derived from myc exon Propertiesofthec-mycprotein I-CAT hybrid genes which demonTwo talks were given on the cellustrated that the first myc exon does lar localization of the c-myc protein, not confer instability to a CAT trans- The c-mycpolypeptide had previouscript ( M Piechaczyk, Montpellier). ly been isolated along with the nucTranslocated myc loci in African- lear matrix after treatment of nuclei derived and sporadic American- with DNase at 37°C. G. Evan (Camderived BL were compared and bridge) presented convincing evifound to activate myc by different dence that the association between mechanisms that may be related to the c-myc polypeptide and the matthe stage of B-cell differentiation. rix is merely a consequence of the Most cases of sporadic BL secrete isolation procedure. Extraction of IgM and contain rearranged myc loci nuclei in isotonic salt medium at 4°C that generally contain truncated first results in the complete recovery of exons. In contrast, primary isolates of c-mycfrom the nucleus with or withAfrican BL contain exon I mutations out priorDNase treatmentwhile diand do not secrete immunoglobulin valent cations and chelators do not (Dalla-Favera). In addition, Burkitt elute c-myc from nuclei. However, lines expressing higher levels of myc myc associates with an insoluble were more tumorigenic in nude mice complex in heat shocked cells that and low level myc BL converted to a also contains HSP 70, p53 and the high level myc expressor by DNA SV40 large T protein. The half life of transfection became more tumori- the myc polypeptide is also engenic. hanced upon heat shock. The n-myc The involvement of immunoglobu- and c-myc polypeptides possess similin gene recombination sequences in lar properties. Tumors with amplified chromosome translocations was de- n-myc or c-myc loci contain proscribed for different neoplasias. portionately higher levels of the myc Strong evidence for the role of JK polypeptide implying that a larger heptamer and nonamer recognition steady state pool of myc protein sequences was provided for a BL 2;8 results from these amplifications variant translocation that occurred (Evan). Cells microinjected with bacmore than 5 kb 3' of c-myc but terially produced human c-myc proinvolved a human Alu repeat con- tein incorporate [3H] thymidine taining Ig recombination sequences when cultured in platelet-poor plason a chromosome 8 (M. Lipp, ma (PPP)suggesting that it may rep-

and tteatures-- ]

resent a competence factor (R. Watt, Philadelphia). Antibody to c-mycwas found to inhibit DNA but not RNA synthesis. Microinjected myc sequesters into a particulate fraction within the nucleus which becomes diffuse upon treatment with actinomycin D. Independent experiments with antiRNP and anti-myc sera revealed a remarkably analogous particulate staining pattern in v-myc expressing 08 cells and colo 320 cells (Watt). The mechanism(s) by which c-myc and other types of nuclearassociated 'immortalizing oncogenes' contribute to the transformed phenotype remains unknown. C. Cerni (Vienna) presented evidence that the c-myc, v-myc, E1A and polyoma large T gene products may contribute to enhanced sister chromatid exchange frequencies in cultured rat lines while transforming oncogenes like ras and polyoma middle T do not. The effect of the immortalizing oncogene on sister chromatid exchange would appear to be recessive since cells that are harboring both classes of activated oncogene displayed low frequencies of exchange. Such extreme variations in recombinagenic activity suggest that the physiological state induced by immortalizing and transforming oncogenes is radically different. Perhaps the deregulated expression of a myc class oncogene can result in progressive changes which may even adversely effect cell viability unless a second oncogene is also subsequently activated to yield a fully transformed phenotype. One conclusion from the meeting was that the c-myc gene and its product hold untold secrets about the biology of cell proliferation. The relevance of the biology of the c-myc gene and its product to natural and induced forms of B-cell neoplasia is compelling to say the least. We can only be pleased to see the growth and development of this exciting field. The papers from this meeeting will be published in a forthcoming edition of Current Topics in Microbiology and Irnmunology. KennethMarcu is at the University of New Yorkat Stony Brook, NY 11790, USA; Fritz Melchersis at the Basellnstitute for Immunology, CH-4058 Basel, Switzerland; Herbert Morse is at the National Institute of Aflergy and InfectiousDiseases;and MichaelPotteris at the NationalCancerlnstitute, Bethesda,MD 20205, USA

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