B cells malignantly transformed by human immunodeficiency virus are polyclonal.

The roles that human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) play in the genesis of acquired immune deficiency syndrome (AIDS)-related lymphomas are not understood. A human B cell line (B-HIV), developed to study AIDS-related lymphomagenesis, contains EBV and HIV genomes and is malignantly transformed. This line was produced by exposing B cells from an EBV-seropositive donor to HIV. To investigate the number of independent transformation events that took place at the time of HIV infection, we sought to determine how many transformed lineages are present in this cell line. B-HIV was found to have multiple different sites of HIV integration (> or = 25) as determined by fluorescence in situ hybridization. As a control, we analyzed a clonal cell line of HIV-infected human T cells, 8E5, and found HIV sequences located exclusively at band q22 in chromosome 13. We conclude that B-HIV is polyclonal, and viral sequences are located at multiple variable chromosomal sites in different B-HIV cells.

A model system for regulation of chronic HIV-1 infection in peripheral B lymphocytes.

B-HIV1, an oligoclone of immortalized cells derived from human peripheral B lymphocytes infected in vitro with the TIIIB isolate of HIV-1, produces low levels of replication-competent HIV when propagated in 1% serum, but increases production > or = 5-fold after phorbol myristate acetate (PMA) exposure. Electron microscopy reveals budding of mature virions from the plasma membrane, without concentration in endocytotic spaces. The PMA effect is specific for protein kinase activation, occurring upon exposure of B-HIV1 to those congeners capable of upregulating calcium and phospholipid dependent protein kinase C and susceptible to inhibition by the protein kinase antagonists H-7 and staurosporine. Induction could also be effected by another viral activator, 5-azacytidine, which acts via an alternate mechanism, and blocked by high doses of interferon-alpha but not the anti-viral nucleoside analog zidovudine (AZT). B-HIV1 may provide a model system for study of the regulation of chronic HIV infection in cells of B lymphocyte lineage.

Human immunodeficiency virus activates c-myc and Epstein-Barr virus in human B lymphocytes.

We have established a line of malignantly transformed human B cells by infecting purified primary B lymphocytes with human immunodeficiency virus type 1 (HIV-1). This line, termed B-HIV1, may serve as a model system for a subset of AIDS-related B-cell lymphomas in which the transformed phenotype may be initiated and/or maintained through an HIV-1 gene product. The B-HIV1 line contains both Epstein-Barr virus (EBV) and HIV-1 genomes. In addition, the c-myc gene is expressed at levels 10 to 20 times those in normal B cells. Similarly, EBV sequences, including those for the latent membrane protein (LMP), are expressed at greatly enhanced levels relative to expression in normal, EBV-immortalized B cells. The upregulation of c-myc and of EBV gene expression can both be produced by infection of susceptible B cells (not already harboring HIV) with exogenous HIV-1. The B-HIV1 line exhibits properties of malignantly transformed cells, in that it grows logarithmically in 1% serum, clones in soft agar, and produces invasive, malignant B-cell lymphomas in severe combined immunodeficient (SCID) mice. We have shown that HIV-1 has the ability to infect primary human B cells and to activate expression of EBV and c-myc. HIV activation of EBV has been documented previously in certain cell lines, here we note that such activation can occur in primary B cells and, under certain conditions, can result in outgrowth of immortalized cell lines. This phenomenon may contribute to the clinical manifestation of lymphadenopathy early after infection with HIV. In addition, we have demonstrated that HIV infection of primary B cells in vitro can result in appearance of a fully malignant phenotype. This phenotype is likely to be due, at least in part, to the activation of c-myc by HIV. Preliminary experiments indicate that Tat, the gene product of the transactivator of viral gene transcription tat, can upregulate c-myc transcription after addition to the culture media of certain B-cell lines. This raises the possibility that Tat can bind to target sequences in cellular RNA and enhance transcription as it does for HIV.(ABSTRACT TRUNCATED AT 400 WORDS)

Suppression of deregulated c-MYC expression in human colon carcinoma cells by chromosome 5 transfer.

Two-thirds of sporadic colon carcinomas express elevated levels of the c-MYC protooncogene. In addition, most colon carcinoma cell lines show constitutive elevated expression (10- to 40-fold over normal) of MYC RNA and protein that is not modulated in response to a mitogenic stimulus. Indirect immunofluorescence has been used to detect c-MYC protein in such cell lines, in hybrid cells resulting from fusions of such lines with cells that regulate MYC normally, and in carcinoma cells to which a normal copy of chromosome 5 has been transferred by microcell fusion. The deregulated expression of c-MYC is suppressed by fusion with a cell that regulates MYC normally. In addition, transfer of chromosome 5 by microcell fusion results in suppression of deregulated expression. Suppressed cells are no longer tumorigenic in nude mice. Loss of the transferred chromosome results in reexpression of the tumorigenic phenotype and in constitutive elevated expression of MYC. These data indicate that function of a tumor-suppressor gene on chromosome 5 is necessary for the regulated expression of MYC in at least some colon cells. Loss of this suppressor results in deregulated MYC expression and is a necessary, but most likely not sufficient, event for the expression of the tumorigenic phenotype in a subset of colon carcinomas.

Does HIV infection of B lymphocytes initiate AIDS lymphoma? Detection by PCR of viral sequences in lymphoma tissue.

Individuals infected with HIV (Human Immunodeficiency Virus) frequently develop B cell non-Hodgkins lymphoma. Although previous studies have failed to document the presence of HIV sequences in these tumors, the recent demonstration of malignant transformation of primary B lymphocytes by HIV-1 has prompted us to reinvestigate this issue. We have examined DNA extracted from 7 lymphomas and 5 lymphadenopathy specimens for HIV LTR (long terminal repeat), gag, and tat sequences using the polymerase chain reaction (PCR). All samples produced products of the expected size with primers for these regions, indicating the presence of HIV proviral sequences in these tissues. The amount of provirus in the tissue was estimated by normalizing the amount of HIV product to the amount of product for the cellular myc gene or beta globin gene. Products were quantitated during the exponential phase of DNA accumulation. These studies indicated that provirus was present at approximately one copy per cell in the 7 lymphoma samples and in 4 of the 5 lymphadenopathy samples. These results are consistent with a direct role for virus in the initiation of lymphoma. Studies to determine whether provirus resides in the lymphoma cells per se will be necessary to further substantiate this hypothesis.

Are there molecular targets for therapy of colon cancer?

Recent advances in understanding the molecular etiology of colorectal carcinoma have made possible a discussion of molecular targets for therapy of the disease. The genes for two inherited predispositions, familial adenomatous polyposis and the Lynch syndrome, have been mapped to specific chromosomal locations. At least five of the genes that are altered in structure or expression to give rise to the tumorigenic phenotype have been isolated by molecular cloning: The K-ras and N-ras protooncogenes have been shown to be altered by point mutation, and expression of the c-myc protooncogene is deregulated. The p53 and DCC genes, both tumor suppressor genes or antioncogenes, are deleted or altered so as to be rendered nonfunctional. Although a single tumor may not suffer all these changes, available evidence indicates that most colorectal tumors contain at least one of these alterations. In addition, work in cell culture and animal systems indicates that tumor cells may be converted to nonmalignant cells by reversing the effects of just one of these changes.

Human immunodeficiency virus induction of malignant transformation in human B lymphocytes.

Aggressive B-cell lymphomas are occurring with increasing incidence among individuals infected with human immunodeficiency virus (HIV). Several lines of evidence implicate both Epstein-Barr virus (EBV) and c-myc activation in the pathogenesis of a major subset of these tumors. These observations prompted our investigation of interactions among EBV, c-myc, and HIV in primary B cells. We show that nonimmortalized peripheral B lymphocytes from EBV-seropositive, HIV-seronegative donors can be infected by HIV and that a subset of these lymphocytes become transformed. Malignant transformation was documented by several criteria. These cells displayed altered growth properties, propagating in 1% serum and cloning in soft agar, and formed invasive tumors of Burkitt lymphoma phenotype after subcutaneous injection into severe combined immunodeficiency mice. Such cells revealed marked enhancement of EBV DNA and RNA and of endogenous c-myc transcripts and protein. HIV-1 infection of already immortalized B-cell lines led to a similar upregulation of EBV and c-myc transcripts. These data indicate that HIV has properties of a transforming retrovirus, as it mediates two events linked to B-cell neoplasia: deregulation of c-myc and activation of EBV. They also raise the possibility of a role for HIV, apart from induction of immune suppression, in the pathogenesis of B-cell lymphoma in the acquired immune deficiency syndrome.

Evidence that the familial adenomatous polyposis gene is involved in a subset of colon cancers with a complementable defect in c-myc regulation.

Human colorectal carcinomas frequently express elevated levels of c-myc mRNA in the absence of a gross genetic change at the c-myc locus. To test the hypothesis that these tumors are defective in a gene function necessary for the regulation of c-myc expression, we fused an osteosarcoma cell line that exhibits normal c-myc regulation with two colon carcinoma cell lines that express deregulated levels of c-myc mRNA. The levels of c-myc transcripts in all of the hybrid clones examined were normal and were induced normally by a mitogenic stimulus. Since rates of c-myc mRNA turnover in the colon carcinoma cells were found to be comparable to those in normal cells, increased message stability cannot account for the increased steady-state levels of transcripts. Our findings suggest that loss of function of a trans-acting regulator is responsible for the deregulation of c-myc expression in a major fraction of colorectal carcinomas. Analysis of restriction fragment length polymorphisms in tumor/normal tissue pairs from patients with primary colorectal lesions indicated that deregulation of c-myc expression in the tumors is correlated with frequent loss of alleles of syntenic markers on chromosome 5q; allele loss on 5q could be detected in 9 of 19 tumors expressing deregulated levels of c-myc mRNA, but not in any of 8 tumors expressing normal levels of c-myc RNA. Chromosome 5q is the region known to contain the gene for familial adenomatous polyposis, an inherited predisposition to colon cancer. These findings, together with the earlier finding that the colonic distribution of tumors exhibiting deregulated c-myc expression is similar to that reported for familial polyposis, provide evidence that loss of function of the familial adenomatous polyposis gene is involved in a subset of colorectal cancers in which c-myc expression is deregulated.

Somatic mutation and transcriptional deregulation of myc in endemic Burkitt's lymphoma disease: heptamer-nonamer recognition mistakes?

We examined the structure and expression of the myc protooncogene in DNA extracted from a primary (uncultured) endemic Burkitt's lymphoma sample designated eBL3. Dot and Northern (RNA) blot analyses demonstrated extreme levels of myc RNA in the eBL3 sample. Nearly complete sequence data of the altered myc locus isolated from eBL3 DNA demonstrated extensive mutations (duplications, insertions, and deletions) in critical myc regulatory regions. Taken together, the data support the idea that myc transcriptional deregulation in Burkitt's lymphoma disease may be a consequence of the position and number of mutations produced within and around the myc locus. Furthermore, the myc exon-1-intron-1 hypermutable PvuII site is part of a potential heptamer-nonamer recognition sequence, suggesting a mechanism for mutation in endemic Burkitt's lymphoma disease.

Insertional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma.

The proto-oncogene c-myc is the cellular homologue of the transforming sequence carried by the avian myelocytomastosis virus MC29. A growing body of evidence implicates structural and functional alterations in and around proto-oncogenes such as c-myc in tumorogenesis. Here we report that comparison of the structure of myc from a ductal adenocarcinoma of the breast and from normal breast tissue of the same patient (Sc) revealed a tumour-specific rearrangement of one myc locus and amplification of the other myc locus. (For myc reviews see refs 1-4; for myc involvement in breast neoplasia see refs 5-7.) Within the second intron of the rearranged locus was a non-myc sequence with nearly complete homology to a long interspersed repetitive element (a LINE-1 sequence or L1). In this case, the L1 sequence has functioned as a mobile genetic element to produce a somatic mutation.

The c-myc protein is constitutively expressed at elevated levels in colorectal carcinoma cell lines.

In this study, we have employed both indirect immunofluorescence and ELISA assays to compare the relative levels of c-myc protein in cell lines derived from normal human colon and colon adenocarcinomas. We show that the levels of protein found in the majority of carcinoma cell lines are consistent with the levels of mRNA expressed, and that both are significantly elevated with respect to the levels found in normal cells. Growing populations of fibroblastic and epithelial cell lines derived from normal colonic mucosa exhibit small numbers of steady-state transcripts and immunofluorescence signals which are weak and confined to the nucleus. The adenocarcinoma cell lines, however, express 5- to 10-fold elevated levels of c-myc mRNA and exhibit correspondingly intense immunofluorescence signals which appear to reside principally in the nucleus. Quantitation of c-myc protein levels in these tumor cell lines by ELISA assay indicates that they are 8- to 37-fold higher than the levels of protein in normal cells. Elevated expression of the c-myc gene at both the mRNA and protein levels occurs constitutively in the colorectal carcinoma cell lines during their growth in culture, in contrast to the transiently elevated levels of expression observed in normal cells which have been subjected to a mitogenic stimulus. The constitutively elevated expression of the c-myc protein in colorectal carcinoma cell lines is not typically accompanied by gross rearrangement or amplification of the gene.

Noncorrelation of the expression of the c-myc oncogene in colorectal carcinoma with recurrence of disease or patient survival.

Our previous work has shown that 26 of 38 cases (68.4%) of primary adenocarcinoma of the colon exhibited significantly elevated levels of c-myc RNA compared to normal colonic mucosa (M. D. Erisman, P. G. Rothberg, R. E. Diehl, C. C. Morse, J. M. Spandorfer, and S. M. Astrin. Mol. Cell. Biol., 5: 1969-1976, 1985; P. G. Rothberg, J. M. Spandorfer, M. D. Erisman, R. N. Staroscik, H. F. Sears, R. O. Petersen, and S. M. Astrin. Br. J. Cancer, 52: 629-632, 1985). In this study, we have compared those levels of expression to the clinical profiles of the affected individuals in an effort to define useful correlates, especially with regard to recurrence of disease and patient survival. Log-rank comparisons of recurrence curves for the entire patient population, for those patients with low levels of c-myc RNA in resected tumor tissue, and for those patients with significantly elevated levels of RNA show no statistically significant differences. Similarly, no statistically significant difference was observed between the high- and low-myc RNA groups with respect to their survival during the postoperative period of observation (median, 25 months). Consequently, the levels of c-myc gene expression observed in primary tumor tissue did not help to define those individuals at higher or lower risk for recurrence of disease and did not point to the likelihood of increased or decreased survival in individuals undergoing surgery for adenocarcinoma of the colon.

Enhanced expression of the c-myc gene in bovine leukemia virus-induced bovine tumors.

We have detected elevated levels of c-myc gene expression in neoplastic cells from all seven bovine leukemia virus (BLV)-induced bovine tumors examined, but not in BLV-infected, nonneoplastic lymphoid cells. No rearrangement or amplification of the c-myc gene could be demonstrated in any of the BLV-induced tumors. Furthermore, BLV proviral DNA was found to have no preferred site of integration in these tumors. The possible mechanisms of enhanced expression of the c-myc gene in BLV-induced tumors have been discussed.

Deregulation of c-myc gene expression in human colon carcinoma is not accompanied by amplification or rearrangement of the gene.

The structure and expression of the c-myc oncogene were examined in 29 primary human colon adenocarcinomas. Dot blot hybridization of total RNA showed that 21 tumors (72%) had considerably elevated expression of c-myc (5- to 40-fold) relative to normal colonic mucosa. These data were corroborated by Northern blots of polyadenylated RNA, which showed a 2.3-kilobase transcript. Southern analysis of the c-myc locus in these tumors indicated the absence of amplification or DNA rearrangement in a 35-kilobase region encompassing the gene. In a parallel study, elevated expression of c-myc without amplification or DNA rearrangement was also observed in three of six colon carcinoma cell lines examined; in addition, unlike a normal colon cell line control, these three cell lines exhibited constitutive, high-level expression of the gene during their growth in cultures. These results indicate that elevated expression of the c-myc oncogene occurs frequently in primary human colon carcinomas and that the mechanism involved in the regulation of c-myc expression is altered in tumor-derived cell lines.

Structure and expression of the oncogene c-myc in fresh tumor material from patients with hematopoietic malignancies.

c-myc is the cellular gene homologous to the transforming sequence of MC29, an acute avian retrovirus. The human c-myc gene was cloned and used to study the structure and expression of c-myc in a variety of human hematopoietic malignancies. In a careful study of 106 patients, c-myc RNA was found to be expressed at elevated levels in tumor cells of 17 leukemia patients and five lymphoma patients. The c-myc gene was found to be rearranged in two lymphomas, an African Burkitt's lymphoma and a non-Hodgkins lymphoma in leukemic phase. The Burkitt's rearrangement involved the insertion of new DNA sequences upstream from the c-myc 5' coding region, presumably replacing the normal c-myc transcriptional promoter. None of the other 104 patients, including 20 with elevated myc expression, exhibited any evidence of a genetic rearrangement involving the c-myc gene. Our results show that there is a subset of hematopoietic malignancies characterized by elevated expression of c-myc. This elevated expression in most cases is not due to obvious genetic changes (rearrangement, amplification) at the c-myc locus nor to chromosomal translocations in the vicinity of this gene.

The role of promoter insertion in the induction of neoplasia.

It has been shown in several retroviral systems that proviral DNA can integrate into the host genome in such a manner that expression of a nearby oncogene is enhanced. This enhancement results from either a direct promotion of transcription from a strong promoter within the proviral 3' LTR or from less well defined activation in which sequences known as 'enhancers' mediate an increase in the transcription of nearby genes. As a result of this observation, potential oncogenes can now be found by identifying genes whose activity is modulated by the nearby insertion of transcriptional activating elements during oncogenesis. It has also been shown that the genome of a retroviral-like IAP can similarly become integrated adjacent to an oncogene and produce an increase in transcription of that gene. Other examples of possible nonviral promoter insertion events that take place in the oncogenesis of the human Burkitt's lymphoma are discussed elsewhere in this volume.

Regulation of myc gene expression in HL-60 leukaemia cells by a vitamin D metabolite.

HL-60, a cell line established from a patient with promyelocytic leukaemia, responds to a variety of inducing agents by ceasing division and acquiring some of the characteristics of either granulocytes or monocytes. Among the agents so far tested, only a comparative few occur naturally in vertebrates and would appear to have significant clinical potential in the treatment of leukaemic patients. One of the most promising of these is the dihydroxymetabolite of vitamin D3, 1,25(OH)2D3. This compound circulates in normal man and has a major role in calcium homeostasis. Moreover, it has recently been reported that 1,25(OH)2D3 increases the survival time of mice injected with myeloid leukaemia cells. We and McCarthy et al. have previously shown that HL-60 cells respond to near physiological levels of 1,25(OH)2D3 by rapidly acquiring a number of monocyte-like features. Here we document that these phenotypic changes are preceded by a marked decrement in the expression of the c-myc oncogene. In fact, the diminution in the level of c-myc mRNA parallels the dose dependency and metabolite specificity shown by the various other indicators of phenotypic change. In addition, we demonstrate that removal of vitamin D3, after the onset of maturational change, results in the reappearance of elevated myc mRNA levels. We believe this to be the first demonstration of a sequential relationship between the application of an exogenous inducing agent, a reduction in myc mRNA levels and the development of characteristics associated with normal cell maturation.