Relationship between tumorigenicity and the dosage of lymphoma- vs. normal-parent-derived chromosome 15 in somatic cell hybrids between lymphoma cells with rearranged pvt-1 gene and normal cells

Somatic cell hybrids were generated between YACUT, a doubly drug-resistant subline of YAC-1 (a Moloney-virus-induced T-cell lymphoma of strain A/Sn origin with 2 proviral insertions near the pvt-1 locus) and normal diploid fibroblasts of CBAT6T6 origin. Three independent fusions were performed. Three uncloned hybrid cultures and 9 independently-derived clones were tested for tumorigenicity by the inoculation of graded cell numbers into syngeneic hosts. One of 3 uncloned hybrid cultures and 3 of 9 clones were weakly tumorigenic (take incidence 0%), and 1 of 3 uncloned hybrid cultures and 6 clones were highly tumorigenic (take incidence greater than 80%). One weakly tumorigenic hybrid and 3 weakly tumorigenic clones carried 3 copies of the tumor-derived chromosome 15 and 2 copies of the normal fibroblast-derived t(14;15) chromosomes. In contrast, 2 highly malignant hybrid clones lost one copy of the normal-fibroblast-derived t(14;15), but contained increased numbers (3.44-4.44) of the tumor-derived chromosome 15. Four tumorigenic segregants selected from the weakly tumorigenic fibroblast hybrids by in vivo inoculation showed the same cytogenetic change as the highly tumorigenic hybrid clones, in that the ratio of the normal:tumor-derived chromosomes 15 changed from 1.18-1.55 to 4.11-5.71. Tumorigenicity was thus associated with a modified balance between the tumor vs. the normal-parent-derived 15-chromosomes. Instead of the usual 3:2 ratio, the tumor-derived 15-chromosomes increased disproportionately, whereas the relative number of the normal-parent-derived 15-chromosome decreased, as a rule. These results suggest that amplification of the lymphoma-derived chromosome 15 favors tumorigenicity, but that this effect is counteracted by some influence emanating from the normal-parent-derived homologous chromosome.     

Correlation of myc expression with the growth-arrested and transformed phenotypes in hybrids between a T lymphoma and an antigen-responsive T-cell line.

Fusion of the YACUT lymphoma cell line with the Mls-1a-antigen-specific non-tumorigenic T-cell line G4 produced growth-arrested hybrids that could be induced to proliferate in the presence of Mls-1a antigen. Prolonged growth of such hybrids by repeated antigenic stimulation resulted in the appearance of autonomously growing hybrid lines. Of the 4 antigen-independent hybrid clones, I was weakly tumorigenic (25% incidence) while the other 3 were highly tumorigenic (100% incidence). In the growth-arrested hybrids the de-regulated c-myc expression characteristic of the YACUT cells was suppressed. In the autonomously growing clones, however, c-myc expression had reverted to the levels of the lymphoma parent and 1 to 2 extra copies of chromosome 15 were consistently present. These results indicate that repeated antigenic stimulation somehow abrogated the down-regulation of c-myc in the growth-arrested hybrid lines. The increase in the number of copies of chromosome 15, however, suggests that genes located on this chromosome may abolish the effect of the negative regulatory functions of the non-malignant parent in a gene-dosage-dependent manner.     

Modification of myc gene amplification in human somatic cell hybrids

In order to study whether cell fusion would modify the DNA copy number of an amplified oncogene, somatic cell hybrids were made between the human neuroepithelioma cell line MCIXC and HeLaCOT human adenocarcinoma cells. MCIXC contains approximately 21 copies of the c-myc oncogene and HeLaCOT contains approximately 5 copies relative to the control. All hybrid clones investigated displayed a marked decrease in the number of copies of c-myc DNA (an average of 5 copies), while the level of c-myc RNA in the hybrids was similar to that found in both parents. All eight hybrid clones were found to be completely nontumorigenic even though both parent cells formed tumors in 100% of the nude mice treated by injection. This loss of oncogene amplification in the hybrids was shown not to be due to either heterogeneity of c-myc amplification in the MCIXC parent or segregation of a copy of the chromosome 22 from the hybrids. This loss most likely resulted from the breakdown of a homogeneously staining region (containing the amplified gene copies) into double minutes, which were subsequently lost from the cells. The HeLaCOT cell line was also fused to the human neuroblastoma BE(2)C, which contains approximately 123 copies of the N-myc oncogene relative to control. Ten hybrid clones were found to contain an average of 47 copies of N-myc DNA, significantly less than the 91 copies predicted had no loss occurred. These BE(2)C x HeLaCOT hybrids expressed on average about 15% the N-myc RNA seen in the BE(2)C parent and, as with the MCIXC x HeLaCOT hybrids, were found to be completely nontumorigenic. However, upon passage in culture, one BE(2)C x HeLaCOT hybrid eventually became tumorigenic. This hybrid also displayed reduced copies of N-myc DNA in comparison to its parent hybrid but surprisingly showed a 2-fold increase in N-myc RNA. Thus, the expression of N-myc, but not the amplification state of either myc gene, appears to correlate with the tumorigenicity of the cells.     

In vivo amplification and rearrangement of c-myc oncogene in human breast tumors

We have studied the genomic organization of cellular myc (c-myc) proto-oncogene in 48 human primary breast tumors. Two types of alterations (amplification and rearrangement) were observed in 27 (56%) of the tumors studied. The c-myc proto-oncogene appeared to be amplified 2- to 15-fold in the DNA of 20 tumors (41%). Non-germ line c-myc-related fragments (rearrangements) of variable size were detected in 7 primary breast tumors (6 malignant, 1 benign); 4 of these tumors presented both rearrangement and amplification, and the other 3 presented rearrangement only. The majority of the tumors analyzed were invasive ductal adenocarcinomas; 58% of these showed c-myc locus genetic alterations. Although the c-myc alterations described here do not appear to correlate with the aggressive behavior of primary breast tumors, they seem to be associated with development of breast carcinoma.     

Cessation of autonomous proliferation of mouse lymphoma EL4 by fusion with a T cell line

Benzanthracene-induced C57BL/6 (H-2b) mouse T-cell lymphoma EL4 (a thymidine kinase-deficient cell line) was fused by using polyethylene glycol with an Mlsa (Mls for minor lymphocyte stimulatory) antigen-dependent T cell line, which was designated G4 and had been derived from a C3H/He mouse (H-2k), and the fused cells were cultured in HAT medium. Although no growing cells appeared in most of these fusions, we consistently obtained growth-arrested H-2Kb-positive cells from the fused cell populations by the panning method. The cells were tetraploid and were able to proliferate in response to Mlsa antigen. Three H-2Kb-positive clones, isolated by limiting dilution from three different fusions, were shown to be EL4 x G4 hybrids, because (1) they had both H-2k and H-2b antigens; (2) each of the clones had one submetacentric chromosome which was a marker chromosome of EL4, and they were tetraploid with modal chromosome numbers of 74, 78, and 79, respectively; (3) they had 4 isozymes of both parental cells. These results indicate that EL4 lymphoma cells cease to proliferate when fused with T cell line G4. The malignant phenotype of lymphoma EL4 is thus suppressed at the level of cell transformation by the introduction of the G4 cell genome.     

Rearrangement of c-myc, pim-1 and Mlvi-1 and trisomy of chromosome 15 in MCF- and Moloney-MuLV-induced murine T-cell leukemias

Provirus insertion near the c-myc, pim-1 or Mlvi-1 genes occurred in 7 out of 59 virally induced T-cell leukemias. C-myc was exclusively rearranged in approximately 10% of MCF247-induced tumors while Mlvi-1 was rearranged to a similar frequency in Moloney-virus-induced lymphomas. Out of 25 karyotyped tumors, 9 (36%) showed trisomy of chromosome 15. Provirus insertion near c-myc, pim-1 or Mlvi-1 occurred both in diploid lymphomas and in tumors with trisomy 15. These results suggest that the molecular and cytogenetic changes observed in murine T-cell leukemias are independent tumor-associated events and that trisomy of chromosome 15 is a common tumor-progression-related event.     

Triplication of one chromosome No. 15 with an altered c-myc containing EcoRI fragment and elimination of the normal homologue in a T-cell lymphoma line of AKR origin (TIKAUT)

An ouabain- and thioguanine-resistant subline (TIKAUT) of spontaneous AKR lymphoma, TKA, was trisomic for chromosome 15 and contained a single 33 kb EcoRI fragment, containing the oncogene c-myc. The original TKA lymphoma and derived in vitro line contained the same 33 kb fragment, as well as a normal 22 kb fragment. It has been concluded that the original 15-trisomic TKA tumor has duplicated a 15-chromosome that contained the changed fragment, while maintaining the normal fragment as well. Subsequently, in the derived TIKAUT line, the changed chromosome duplicated again, giving rise to three copies, and the normal homologue was eliminated altogether. This confirms our earlier somatic hybrid study showing that the duplicated 15-chromosome of a T-cell leukemia confers an advantage on the cell that favors tumorigenicity, whereas the normal homologue exerts a counteracting influence. Therefore, in the course of tumor progression, the changed chromosome tends to be amplified, whereas its normal homologue tends to be eliminated.     

Rearrangements of bcl-1, bcl-2, bcl-6, and c-myc in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphoma (DLBL) is characterized by a marked degree of morphologic and clinical heterogeneity. We studied 137 patients with de novo DLBL for rearrangements of the bcl-1, bcl-2, bcl-6 and c-myc oncogenes by Southern blot analysis. Structural alterations of bcl-1, bcl-2, bcl-6, and c-myc were detected in 21 of 137 (15.3%), 8 of 137 (5.8%), 22 of 137 (16.1%), 14 of 137 (10.2%) patients, respectively. Two cases showed a combination of bcl-1 and bcl-6 rearrangements. Chromosomal analysis was performed in 31 cases of the 137 DLBL. 27 of these showed karyotypic abnormalities, and two had translocations 3q27 involving bcl-6. However, one of two cases had no rearrangement of bcl-6. Patients with rearranged bcl-6 and c-myc tended to have poorer survival than patients with germ-line. Furthermore, bcl-1 and bcl-2 rearrangements tended to have a better outcome, although the above differences were not statistically significant. Rearrangements of the bcl-1, 2, 6, and c-myc gene correlated with the clinical outcome in DLBL and may thus serve as prognostic markers in patients with this form of malignant lymphoma. However, other genetic factors are probably involved in determining prognosis.     

Suppression of the translocated myc gene and expression of intracisternal A-particle genes in tumorigenic and non-tumorigenic hybrids between murine myeloma and normal fibroblasts

We have studied the tumorigenic potential of a series of independent intraspecies hybrid clones derived from fusion of murine myeloma (BALB/c) and normal fibroblasts (C3H). All of these hybrids grew as adherent cells and thus resembled the fibroblast phenotype. As judged by chromosome enumeration, these hybrids appear to retain the full complement of their parental cells. Three out of 4 hybrids tested were able to form colonies in soft agar and to grow as tumors in either nude or (BALB/c x C3H) F1 mice, albeit at a reduced rate. The 4th hybrid did not grow in agar, was non-tumorigenic and may have had a 2:1 fibroblast to myeloma genomic equivalence ratio. In contrast to the parental myeloma cells, all the hybrids exhibited restricted growth rates in serum-free medium. As in our previous sets of hybrids formed between myeloma and L-cells, expression of the Ig genes was inhibited in the new hybrids and the derived tumors. The constitutive expression of the translocated myc gene in the myeloma parental cells was decreased in the hybrids and in all their derived tumors. In contrast, all of the hybrid cell lines and the tumors express high levels of the intracisternal A particle mRNAs. Our results show that the tumorigenic phenotype of myeloma cells is either fully or partially suppressed in myeloma x fibroblast hybrids and that this may be due to the fact that expression of the translocated c-myc is suppressed. We suggest that, in addition to the translocated myc gene, myeloma cells contain other activated oncogene(s), and that the latter are responsible for the residual tumorigenic potential of the myeloma x fibroblast hybrids.     

Amplification of c-myc in hepatocellular carcinoma: correlation with clinicopathologic features, proliferative activity and p53 overexpression.

Expression of the proto-oncogene c-myc has been implicated in liver regeneration and hepatocarcinogenesis. The biologic significance of c-myc gene amplification in human hepatocellular carcinoma, however, is unconfirmed. We correlated c-myc gene amplification with clinicopathologic features, proliferative activity, and p53 expression in 42 resected tumors. c-myc amplification in tumor tissue was determined using a differential polymerase chain reaction, a useful procedure for the evaluation of gene amplification in archival formalin-fixed paraffin-embedded tissues, in comparison with a dopamine D2 receptor gene. Proliferative activity was estimated by numbers of argyrophilic nucleolar organizer regions and immunohistochemical nuclear labeling rates using a monoclonal antibody against Ki-67. The c-myc gene was amplified in 14 of 42 tumors (33.3%). Amplification of c-myc was more frequent in younger patients and in larger tumors, and less differentiated tumors. No correlation was noted with alpha-fetoprotein level or viral hepatitis state. The amplification showed positive correlation with both proliferative activity and p53 overexpression. Disease-free survival in patients showing c-myc amplification was significantly shorter than in those without amplification. These results suggest that c-myc amplification is an indicator of malignant potential and poor prognosis in hepatocellular carcinoma. c-myc amplification and p53 alteration may be coparticipating events in the progression of these tumors.     

Tumorigenicity of SEWA murine cells correlates with degree of c-myc amplification

Previous studies have shown that cells of the SEWA mouse tumor contain amplified copies of the proto-oncogene c-myc in the aberrant chromosomal structures of double minutes (DMs), homogeneously staining regions (HSRs) and C-bandless chromosomes (CMs). DMs, and to a lesser degree CMs, tend to disappear from the cells grown in vitro and again reappear after transfer back in vivo, as if DNA amplification confers a growth advantage upon the tumor cells. We have now isolated five in vitro clones that exhibit different degrees of c-myc amplification. When we inoculated cells of the different clones into compatible hosts, we found that there was a positive correlation between degree of c-myc amplification, level of c-myc RNA, and tumorigenicity. Our results lend further support to the idea that gene amplification contributes to the higher malignant phenotype, and to progression of tumors.     

High correlation between molecular alterations of the c-myc oncogene and carcinoma of the uterine cervix

We have examined 35 human tumors of the uterine cervix (carcinoma presenting the highest incidence in Mexico; about 34% of women's malignant tumors) for alterations of the cellular myc (c-myc) protooncogene. Elevated amplification and/or rearrangement of the c-myc oncogene were detected in most (approximately 90%) samples (48% showed amplification and 43% presented both alterations). Most tumors were stage II cervical carcinomas and for some of them we detected up to 60-fold amplification of c-myc. These results suggest an important role for c-myc oncogene in the development of tumors of the uterine cervix.     

Somatic cell hybrids of the Djungarian hamster: malignancy and evolution of the karyotype

Djungarian hamster somatic cell hybrids were obtained by fusing malignant SV40-transformed fibroblasts (line DM15, HGPRT-), and normal male lymphoid cells. Tumorigenicity, growth in soft agar and karyotype changes of 10 independent hybrid clones were studied. All hybrids grew as tumors after injection of new-born hamsters with 1 x 10(6) cells. A total of 313 tumors occurred in 523 hamsters. The hybrids proliferated in soft agar as well. No correlation was noted between the ability of hybrids to grow in vivo and to form colonies in soft agar. The total chromosome number in hybrid cells was usually less than the expected sum of the parental chromosome sets. G-banding analysis showed that, in vitro, hybrids lost chromosomes of the normal parent, whereas marker chromosomes of the malignant parent were retained. In the majority of hybrid tumors the chromosome set was reduced to the diploid range. In tumors with a slightly reduced karyotype one or two homologues of chromosomes #4 and #8 were, as a rule, eliminated.     

Amplification of the c-myc gene in human medulloblastoma cell lines and xenografts

Cultured cell lines and xenografts derived from 7 human medulloblastomas were evaluated for amplification of the c-myc, N-myc, epidermal growth factor receptor, and gli genes by Southern blot analysis. Karyotypes of the original biopsies and early passaged cells demonstrated double minute chromosomes in 4 of the 7 cases. All 7 samples (3 cell lines and 4 xenografts) from the 4 tumors with double minute chromosomes contained amplification of the c-myc gene. Cell lines and xenografts derived from the 3 biopsies without double minute chromosomes failed to demonstrate amplification of the 4 genes which were tested, but a rearrangement of the c-myc gene occurred in 1 of the 3 tumors. These observations demonstrate that the c-myc gene is often amplified and/or rearranged in human medulloblastomas and suggest that amplification of this gene provides a growth advantage for medulloblastoma cells in vitro and in vivo.     

c—myc基因在乳腺癌,膀胱癌和肾癌组织中的扩增研究

目的研究乳腺癌、膀胱癌和肾癌组织中ｃ－ｍｙｃ基因扩增情况。方法采用聚合酶链式反应（ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃ－ｔｉｏｎ,ＰＣＲ）,对４８例恶性肿瘤ｃ－ｍｙｃ基因扩增进行研究,通过激光密度扫描仪对琼脂糖凝胶ＥＢ染色底片进行积分光密度分析。结果正常组织无ｃ－ｍｙｃ扩增,ｃ－ｍｙｃ与ｎ－ｍｙｃ＋１－ｍｙｃ比值９５％可信区间为０．０８６９－０．６２５７,乳腺癌组织ｃ－ｍｙｃ基因扩增阳性率为     

Demonstration of clonal selection in a child with non-Hodgkin's lymphoma

A 2-year-old boy with B-lineage non-Hodgkin's lymphoma is described. He presented with growing skin tumors on his head, and biopsy specimens showed a malignant lymphoma of diffuse lymphoblastic type. Sixty-four percent of bone marrow cells were replaced with lymphoblasts, and they expressed B-lineage markers (CD19 and HLA/DR). Southern blot analysis demonstrated immunoglobulin heavy chain gene rearrangements (two rearranged and one germline) with the germline configuration of the T-cell receptor beta chain gene. Ten months later he relapsed with blasts of M5 morphologic type and a myeloid phenotype with the germline configuration of the immunoglobulin genes. During the next 2 months, myeloid blasts with immunoglobulin gene rearrangement which was identically rearranged with one of the two rearranged bands detected at diagnosis appeared. The most likely explanation for these findings is that initially the patient seemed to have at least two different clones of blasts, and clonal selections occurred during the treatments.