Donald L. Ewert

NSAID-Induced Apoptosis in Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts is Dependent on v-src and c-myc and is Inhibited by bcl-2

Mounting epidemiological and experimental evidence implicates non-steroidal antiinflammatory drugs as anti-tumorigenic agents. Our previous work showed that nonsteroidal antiinflammatory drug treatment of src-transformed chicken embryo fibroblasts caused apoptosis--a mechanism by which these drugs might exert their anti-tumorigenic effect. The present studies employ a sensitive technique for detecting single- and double-stranded DNA cleavage (the comet assay) to quantitate apoptosis. By this method pp60v-src, which antagonizes apoptosis in many cell systems, was found to induce apoptosis in 11-23% of serum-starved fibroblasts. However, treatment with diclofenac following pp60v-src activation produced a much stronger response beginning within 6 hours of treatment that resulted in 100% lethality. During cell death, cyclooxygenase-2 but not cyclooxygenase-1 mRNA was found to be uniformly increased by all apoptotic drugs tested. Examination of the expression of apoptosis-associated genes showed that c-rel and p53 (found in normal or v-src-transformed chicken embryo fibroblasts at moderate levels), and bcl-2 (present at an extremely low level) were largely unchanged by treatment with eight different nonsteroidal antiinflammatory drugs. However, overexpression of human bcl-2 inhibited diclofenac-mediated apoptosis by 90%, demonstrating directly that bcl-2 expression can regulate nonsteroidal antiinflammatory drug induction of cell death. The proto-oncogene c-myc is known to cause apoptosis in chicken embryo fibroblasts when artificially overexpressed in cells deprived of trophic factors. We found that nonsteroidal antiinflammatory drug treatment following pp60v-src activation persistently induced myc protein and mRNA by more than 20-fold above that evoked by pp60v-src activation alone. Moreover, transfection of antisense c-myc oligonucleotides reduced drug-induced myc expression by 80% and caused a concomitant 50% reduction in cell death. These findings suggest that nonsteroidal antiinflammatory drug-induced apoptosis proceeds through a src/myc dependent pathway which is negatively regulated by bcl-2.

Major histocompatibility (B) complex control of the growth pattern of v-src DNA-induced primary tumors

Observations that the major histocompatibility (B) complex is a determinant of the growth pattern of Rous sarcoma virus (RSV)-induced tumors raised the question as to whether control is exerted at the level of a v-src-determined, i.e., transformation-specific, function. To investigate this point, the tumor size scores and tumor profile indices of v-src-induced tumors were compared in two lines of chickens congenic for B complex genotypes. The finding that the growth patterns of tumors, induced by v-src DNA inoculation at 6 weeks posthatch, differ in these two lines establishes that the B complex exerts control over tumor growth at the level of a v-src-determined function. The potential importance of this control, in terms of the naturally occurring case of an avian sarcoma virus infection, is suggested by the observation that the patterns of tumor growth in a given congenic line are similar whether the tumors are induced by v-src DNA or by RSV.

Duck hepatitis B virus is tropic exocrine cells of the pancreas

Earlier observations had established that duck hepatitis B virus (DHBV) is tropic for pancreatic endocrine cells, including cells localized to islets and to acini. Because cells identifiable as endocrine represented only a minor fraction of the total acinar-associated, infected subpopulation, the possibility was addressed in the present study that this subpopulation also comprises exocrine cells. Fixed preparations of cells from pancreas of congenitally DHBV-infected young ducks were reacted in double immunofluorescence assay with anti-virus serum and either anti-avian pancreatic polypeptide (APP) serum, a probe for a major subclass of acinar-associated endocrine cells, or anti-chymotrypsin serum, a probe for exocrine cells. Approximately 2-5% of the cells in these preparations were viral antigen-positive, comprising a minor fraction positive for APP and a much larger fraction positive for chymotrypsinogen. The detection of the latter establishes that DHBV is tropic for exocrine cells.

Blocked B cell differentiation and emigration support the early growth of Myc-induced lymphomas.

Avian leukosis virus induces lymphoma in chickens after proviral integration within the c-Myc gene, and subsequent expansion of Myc-overexpressing lymphocytes within transformed bursal follicles. The clonal expansion of these follicles allowed us to examine how Myc influences cell differentiation, growth, and apoptosis in lymphoid progenitors soon after the onset of Myc overexpression. Immunohistochemical analysis of developmental markers established that Myc overexpression consistently blocks lymphocyte differentiation at a late embryonic stage. Myc-transformed follicles also grow much more rapidly than normal follicles. This rapid growth is not mediated by suppression of apoptosis, as normal and Myc-transformed follicles showed similar rates of cell death by TUNEL immunohistochemical analyis of cells undergoing DNA degradation. Measurements of DNA synthesis and mitotic index showed modest effects of Myc to increase lymphocyte proliferation, as normal lymphocytes already divide rapidly. The major mechanism mediating rapid growth of transformed follicles instead involved failure of myc-overexpressing lymphocytes to emigrate from transformed follicles, while normal lymphocytes actively emigrate after hatching, as measured by BrdU pulse-chase labeling and immunohistochemical measurements. This failure to undergo the normal program of differentiation and subsequent bursal retention of lymphocytes accounts for most of the growth of transformed follicles, while Myc-induced proliferation makes a smaller contribution.

Wing web or intravenous inoculation of chickens with v-src DNA induces visceral sarcomas

We have previously found that sarcomas localized to visceral organs frequently arise following wing web inoculation of Rous sarcoma virus. This observation prompted an investigation of whether visceral sarcomas are also inducible by wing web inoculation of a subgenomic viral DNA fragment (v-src DNA) that includes v-src but lacks genes encoding viral structural protein. For this analysis, line SC chickens were inoculated with v-src DNA at 1-2 days posthatch and monitored for 9 weeks. Ninety percent of the chickens developed wing web (primary) tumors at the site of inoculation and, of these, about 30% exhibited visceral tumors. All tumors were histologically identifiable as sarcomas, and both the primary and visceral sarcoma cells were specifically reactive with two monoclonal antibodies elicited to different peptide fragments of pp60v-src. In a separate set of experiments, visceral sarcomas were also observed in about half of the line SC chickens that had been inoculated intravenously with v-src DNA. These results indicate that exogenous progeny virus production is not required for v-src-induced, visceral sarcoma formation. In addition, they demonstrate that intravenous inoculation of v-src DNA is a means to achieve rapid and widespread, disseminated sarcoma growth.

Induction of a diffuse mesothelioma in chickens by intraperitoneal inoculation of v-src DNA.

The presence of peritoneum-based, cytokeratin-positive tumor cells, as detected under conditions of intravenous inoculation of chickens with a v-src-positive DNA fragment, raised the possibility that such DNA may be inductive for diffuse mesothelioma. To investigate this possibility, chickens were inoculated intraperitoneally with v-src DNA, and the resultant peritoneum-based tumor tissue was subjected to histopathological analysis. On the basis of (i) morphology, (ii) immunohistochemistry/histochemistry, and (iii) ultrastructure, we concluded that this tissue represents a diffuse mesothelioma.

Sarcoma growth in 15l5×72 chickens infected with avian sarcoma viruses of subgroup B or G

A comparative study was made of sarcoma growth in 15I5 x 7(2) chickens infected in the wing web at 4 weeks of age with strains of subgroup B or G avian sarcoma viruses. Infection with sarcoma viruses of either subgroup B or G resulted in the formation of progressive wing web sarcomas at the site of inoculation. The survival times of the subgroup G virus-infected chickens were generally at least twice as great as the survival times of the subgroup B virus-infected chickens, which averaged 6-9 weeks postinoculation. At 5 weeks postinfection, a significantly higher titer of virus neutralizing antibody was detected in the subgroup G virus-infected chickens. Necropsy indicated that a high percentage of subgroup B virus-infected chickens exhibited fibrosarcomas at sites distal to the primary wing web sarcomas, whereas only a small percentage of subgroup G virus-infected chickens exhibited distal sarcomas. The results further indicated that the viral env gene is a determinant of the pattern of distal sarcoma formation.

Expression of endogenous retroviral envelope glycoprotein as a determinant of immunity to rous sarcoma

To analyze the effect of the expression of endogenous retroviral envelope glycoprotein on tumor immunity, patterns of sarcoma growth were compared in inbred FP line chickens infected with either of two strains of avian sarcoma virus, Pr-B (subgroup B) or cl.85 (subgroup G). These viruses were chosen for analysis because the envelope glycoprotein of Pr-B, but not of cl.85, is antigenically cross-reactive with the endogenous retroviral envelope glycoprotein expressed in the FP line. Inoculation of 1-day-old hatchmates with either virus yielded a significant percentage of chickens with distal sarcomas localized to visceral organs. By contrast, a marked difference in the percentage of chickens bearing distal sarcomas was noted when sarcoma tissue excised from virus-inoculated donors was implanted in 1-day-old recipients; a high proportion of the recipients of Pr-B-induced sarcoma tissue (Pr-B-sarcoma recipients), but only a low proportion of the cl.85-sarcoma recipients, exhibited distal sarcomas. At 3 weeks posthatch, a significantly higher percentage of donor-derived cells was detected in the primary tumors of the cl.85- versus the Pr-B-sarcoma recipients. A model of immune control, premised on the tolerogenicity of endogenous viral glycoprotein, is proposed to rationalize these results.

The influence of the ev 3 locus on the inducibility of serum antibody reactivity for envelope glycoprotein group-specific determinants☆

Chickens segregating for the ev 3 locus were bred by backcross matings of line 6(3) to line 15B. Analysis of RAV-1-infected segregants indicated that inducibility of antibody reactivity for envelope glycoprotein group-specific determinants correlated with the absence of ev 3, whereas noninducibility correlated with the presence of ev 3. Since the ev 3(+) and ev 3(-) segregants possessed similar genetic backgrounds, these results provide direct evidence that the ev 3 locus determines the phenotype of noninducibility.

Follicular exclusion of retroviruses in the bursa of fabricius

To gain insight into the regulation of retroviral infection at the cellular level, we analyzed the distribution of retroviral antigen and nucleic acid in the bursa of Fabricius of the parents and progeny of two highly inbred lines of chickens, one resistant and the other susceptible to infection. Line 15I5 chickens and line 7(2), which are C/C and C/A, respectively, and 15I5 x 7(2) F1 chickens were infected with either RAV-1 or RAV-49 avian leukosis virus (ALV). Most bursal follicles of F1 chickens infected with either virus contained a variable mixture of virus-positive and virus-negative cells and a few (1 to 20%) were void of detectable virus. However, in either parental line the respective virus was uniformly expressed among all follicles. The follicles which excluded virus in the F1 birds were indistinguishable from other infected follicles in the same bursa or in uninfected birds on the basis of histology or cellular antigen expression. It was concluded that virus susceptibility is most likely determined at the bursal stem cell level of differentiation, possibly by a process of allelic exclusion at the retroviral receptor locus.