The EV-O-derived cell line DF-1 supports the efficient replication of avian leukosis-sarcoma viruses and vectors.

The lack of a well-behaved permanent, adherent, nontransformed chicken cell line has made some experiments with avian leukosis-sarcoma viruses (ASLV) and vectors considerably more difficult. The EV-O-derived line, DF-1, supports the efficient replication of subgroups (A), (B), and (C) ASLV, as well as amphotrophic murine leukemia virus and an ASLV-derived vector that has its env gene derived from the env gene from an amphotrophic murine leukemia virus. The cell line responds appropriately to the expression of a transforming oncogene (v-myc) to a growth suppressor gene [p21(waf1)] and can be sorted (using FACS) if infected by an ASLV vector that expresses GFP.

Overexpression of p21waf1/cip1 arrests the growth of chicken embryo fibroblasts that overexpress E2F1.

P21waf1/cip1 is a potent inhibitor of cell cycle progression and can inhibit the growth of both normal cells and cells transformed by a number of oncogenes. However, the ability of p21waf1/cip1 to inhibit the growth of cells that overexpress the transcriptional transactivator E2F1 is controversial: it has been reported both that E2F1 can and cannot overcome the block in the cell cycle induced by p21waf1/cip1. To avoid the complications that arise when such experiments are done with permanent cell lines, we tested the effects of overexpressing p21waf/cip1 and E2F1 in primary chicken embryo fibroblasts. In this system very high levels of E2F1 overexpression cause considerable apoptosis; however, the surviving cells still overexpress E2F1. These cells are transformed and their growth is blocked by overexpression of p21waf1/cip1.

Comparative analysis of the structure and function of the chicken c-myc and v-myc genes: v-myc is a more potent inducer of cell proliferation and apoptosis than c-myc.

To gain a more complete understanding of c-myc regulation in chickens, we have completed the structural characterization of the chicken c-myc gene and have begun to investigate c-myc transcription and protein expression. A comparison of c-myc structure and expression between mammals and birds presents an enigma: there are striking similarities in the pattern of gene expression in the absence of obvious sequence similarities in the controlling elements. We have begun to investigate c-myc and v-myc function using retroviral vectors that differ solely in the Myc proteins that they express. We show that while the overexpression of the smaller c-Myc protein is sufficient to induce morphological transformation in chicken embryo fibroblasts, overexpression of v-Myc provides a stronger signal for cells to enter the cell cycle and is a more potent inducer of apoptosis than c-Myc.

Overexpression of human p21waf1/cip1 arrests the growth of chicken embryo fibroblasts transformed by individual oncogenes.

In normal cells, cell growth and division is controlled by the interplay between proto-oncogenes and tumor suppressor genes. Cancer cells usually have both activated an oncogene and have lost a functional tumor suppressor gene. High level expression of a tumor suppressor, p53, can block the growth of cancer cells. waf1/cip1 is transactivated by the tumor suppressor p53 and the p21waf1/cip1 protein is itself a suppressor of cell growth. To test the effect of growth suppression genes on the growth of cells transformed by individual oncogenes, we have used replication-competent retroviral vectors to induce high level expression of p53 and p21waf1/cip1. Overexpression of p21waf1/cip1 arrests the growth of chicken embryo fibroblasts (CEF) transformed by v-Src, tf-Ras, c-Mos and c-Myc. These data suggest that p21waf1/cip1 might be a useful tool in gene therapy for human cancer.

Isolation and characterization of the chicken c-sno gene.

We have cloned and analyzed the chicken c-sno (cellular ski novel) gene. The promoter region and all of the intron/exon boundaries have been sequenced. The gene is approx. 12-kb long and contains six exons, the first of which is noncoding. The amino-acid sequences encoded in this first coding exon of c-sno and c-ski are highly related; however, the remainder of these two genes appears to be unrelated. Although there is evidence that the transcripts of mammalian c-sno are alternatively spliced, there is no evidence that chicken c-sno is alternatively spliced. The promoter region has a high G + C content and contains neither a TATAA nor a CAAT box. Potential binding sites for the transcription factors SP1, AP1 and AP2, are present upstream from the transcription start point.

Bcl-2 expressed using a retroviral vector is localized primarily in the nuclear membrane and the endoplasmic reticulum of chicken embryo fibroblasts.

A complementary DNA for human bcl-2 was cloned into the replication competent avian retrovirus vector RCASBP, and the resulting virus was used to express human Bcl-2 protein at high levels in chicken embryo fibroblasts. The expression of Bcl-2 did not transform or significantly alter the longevity of the chicken embryo fibroblasts in the presence of normal amounts of serum. However, the expression of Bcl-2 blocked c-Myc-induced apoptosis in these cells. Fractionation of the infected chicken embryo fibroblasts indicated that the protein was distributed equally between nuclear and high density cytoplasmic membranes. Immunofluorescence analysis by confocal microscopy and immunoelectron microscopy showed that the Bcl-2 protein was primarily associated with the nuclear membrane and with the endoplasmic reticulum. Reduced amounts of the protein were associated with other membranes in the cytoplasm. These data show that, in this system, the Bcl-2 protein associates with the nuclear membrane and intracytoplasmic membranes but is not preferentially associated with mitochondria.

Retroviruses that express different ras mutants cause different types of tumors in chickens.

We have used replication-competent retroviral vectors to express avian and murine ras genes in cultured chick embryo fibroblasts (CEF) and in chickens. Since the viral vectors are identical, it is possible to compare the oncogenic potential of the ras genes directly. The normal (12 gly) form of chicken c-Ha-ras is not oncogenic in vivo, nor does high-level expression transform CEF. Expression of murine v-ras or modified forms of chicken c-Ha-ras with either lysine or glutamine at position 12 transforms CEF and causes tumors in birds. However, the oncogenic potential of the transforming ras genes is different; the viruses that express the genes with lysine and glutamine at position 12 cause a distinct spectrum of tumors.

Expression of the bcl-2 gene in human multiple myeloma cell lines and normal plasma cells.

The bcl-2 gene, encoding a mitochondrial membrane protein suggested to play an important role in cell survival, is translocated into the Ig loci in about 80% of human follicular lymphomas, which results in a high level of expression. This report shows that bcl-2 was expressed in eight of eight human multiple myeloma cell lines and in normal lymph node and bone marrow plasma cells. In the majority of the myeloma lines, the level of expression was comparable with that observed in Karpas 422, a follicular lymphoma cell line carrying a 14;18 translocation of the bcl-2 gene. DNA rearrangements of the bcl-2 locus were evident in only one of the myeloma cell lines, U-266-1970. In this cell line, which exhibited the highest bcl-2 expression, a fourfold increased copy number of the bcl-2 gene was estimated by Southern analysis. This amplification was lost in cells of later passages (U-266-1984), suggesting that bcl-2 might possibly have played a role in the tumor development in vivo. Our results are in contrast to previous observations in murine plasmacytoma, in which bcl-2 was shown to be silent. The results also contradict the published observation that bcl-2 is not expressed at terminal stages of B-cell differentiation. It is at present unclear whether the high expression of bcl-2 in human myeloma is the result of a deregulated expression associated with the malignant phenotype or a mere reflection of the bcl-2 expression typical of normal plasma cells.

DNA rearrangements in human follicular lymphoma can involve the 5' or the 3' region of the bcl-2 gene.

In most human follicular lymphomas, the chromosome translocation t(14;18) occurs within two breakpoint clustering regions on chromosome 18, the major one at the 3' untranslated region of the bcl-2 gene and the minor one at 3' of the gene. Analysis of a panel of follicular lymphoma DNAs using probes for the first exon of the bcl-2 gene indicates that DNA rearrangements may also occur 5' to the involved bcl-2 gene. In this case the IgH locus and the bcl-2 gene are found in the order 3' C gamma S gamma/mu JH 5'::5' bcl-2 3' (where C = constant, S = switch, and JH = joining segment of the heavy chain locus), suggesting that an inversion also occurred during the translocation process. The coding regions of the bcl-2 gene, however, are left intact in all cases of follicular lymphoma studied to date.