Loss of heterozygosity at the Ink4a/Arf locus facilitates Abelson virus transformation of pre-B cells.

In many tumor systems, analysis of cells for loss of heterozygosity (LOH) has helped to clarify the role of tumor suppressor genes in oncogenesis. Two important tumor suppressor genes, p53 and the Ink4a/Arf locus, play central roles in the multistep process of Abelson murine leukemia virus (Ab-MLV) transformation. p53 and the p53 regulatory protein, p19Arf, are required for the apoptotic crisis that characterizes the progression of primary transformed pre-B cells to fully malignant cell lines. To search for other tumor suppressor genes which may be involved in the Ab-MLV transformation process, we used endogenous proviral markers and simple-sequence length polymorphism analysis to screen Abelson virus-transformed pre-B cells for evidence of LOH. Our survey reinforces the role of the p53-p19 regulatory pathway in transformation; 6 of 58 cell lines tested had lost sequences on mouse chromosome 4, including the Ink4a/Arf locus. Consistent with this pattern, a high frequency of primary pre-B-cell transformants derived from Ink4a/Arf +/- mice became established cell lines. In addition, half of them retained the single copy of the locus when the transformation process was complete. These data demonstrate that a single copy of the Ink4a/Arf locus is not sufficient to fully mediate the effects of these genes on transformation.

Transplantation of virally transduced cells into the dermis of immunocompetent and immunodeficient (SCID) mice to determine gene expression profile and differential donor cell survival.

Cell therapy and bioengineering hold great promise as therapeutic approaches using cells and cell-derived factors to treat various pathologic or trauma-induced states. One possible application is the transplantation of cells into wounded tissue to help regulate tissue repair. Cells engineered for optimal wound healing may help to minimize scarring following surgery or to enhance the rate of healing of chronic wounds. The purpose of the current study was to determine the effect of a viral insert, the LacZ-bearing, first generation adenovirus AdRGD, on the survival of dermally transplanted murine skin allogenic fibroblasts. The LacZ insert facilitated quantitation of both cell survival and gene expression and was used here to measure viable cell number. In addition to bearing the LacZ marker, the AdRGD vector is capable of carrying therapeutic gene inserts, so this study tested the feasibility of gene therapy for wound healing. Murine skeletal muscle PP6 (i.e., Pre-Plate 6) myogenic stem cells served as an alternate donor cell type. Cells were labeled with the LacZ-bearing AdRGD adenovirus vector and injected (50,000 cells/site) into the dorsal skin of adult normal, immunocompetent mice as well as in immunodeficient SCID mice. Skin biopsies were taken on days 0, 1, 2, 3, and 7 post-transplant, and assayed for LacZ expression. Soon after transplant (day 1), cell numbers underwent a transient decrease, but by day 2 post-transplant they were present in appreciable numbers. Between days 2-7 post-transplant, both allogenic fibroblasts and PP6 myogenic stem cells maintained survivability in similar numbers. Further, survival of transplanted cell types was similar in both normal, immunocompetent as well as SCID mice during this time period. There were no signs of acute inflammation or rejection in any of the samples. This study shows that AdRGD-transduced cells are not immunogenic in the mouse skin model and the cells show similar survival for the first 7 days post-transplantation independent of the cell type or immunocompetence of the host.

p53 mediates apoptotic crisis in primary Abelson virus-transformed pre-B cells.

Transformation of pre-B cells by Abelson murine leukemia virus (Ab-MLV) involves a balance between positive, growth-stimulatory signals from the v-Abl oncoprotein and negative regulatory cues from cellular genes. This phenomenon is reflected by the clonal selection that occurs during Ab-MLV-mediated transformation in vivo and in vitro. About 50% of all Ab-MLV-transformed pre-B cells express mutant forms of p53 as they emerge from this process, suggesting that this protein may play an important role in the transformation process. Consistent with this idea, expression of p19(Arf), a protein whose function depends on the presence of a functional p53, is required for the apoptotic crisis that characterizes primary Ab-MLV transformants. To test the role of p53 in pre-B-cell transformation directly, we examined the response of Trp53(-/-) mice to Ab-MLV. The absence of p53 shortens the latency of Abelson disease induction but does not affect the frequency of cells susceptible to Ab-MLV-induced transformation. However, primary transformants derived from the null animals bypass the apoptotic crisis that characterizes the transition from primary transformant to fully malignant cell line. These effects do not require p21(Cip-1), a major downstream target of p53; however, consistent with a role of p19(Arf), transformants expressing mutant p53 and abundant p19 retain wild-type p19 sequences.

p19(Arf) induces p53-dependent apoptosis during abelson virus-mediated pre-B cell transformation.

The Ink4a/Arf locus encodes p16(Ink4a) and p19(Arf) and is among the most frequently mutated tumor suppressor loci in human cancer. In mice, many of these effects appear to be mediated by interactions between p19(Arf) and the p53 tumor-suppressor protein. Because Tp53 mutations are a common feature of the multistep pre-B cell transformation process mediated by Abelson murine leukemia virus (Ab-MLV), we examined the possibility that proteins encoded by the Ink4a/Arf locus also play a role in Abelson virus transformation. Analyses of primary transformants revealed that both p16(Ink4a) and p19(Arf) are expressed in many of the cells as they emerge from the apoptotic crisis that characterizes the transformation process. Analyses of primary transformants from Ink4a/Arf null mice revealed that these cells bypassed crisis. Because expression of p19(Arf) but not p16 (Ink4a) induced apoptosis in Ab-MLV-transformed pre-B cells, p19(Arf) appears to be responsible for these events. Consistent with the link between p19(Arf) and p53, Ink4a/Arf expression correlates with or precedes the emergence of cells expressing mutant p53. These data demonstrate that p19(Arf) is an important part of the cellular defense mounted against transforming signals from the Abl oncoprotein and provide direct evidence that the p19(Arf)-p53 regulatory loop plays an important role in lymphoma induction.

Mutation of Tp53 contributes to the malignant phenotype of Abelson virus-transformed lymphoid cells.

Abelson murine leukemia virus transforms pre-B cells in vitro and induces rapid-onset pre-B-cell lymphoma in vivo. Expression of an active v-Abl protein tyrosine kinase is required for the oncogenic functions of the virus. Despite the strong growth-stimulatory signal provided by v-Abl, the virus-induced tumors are clonal or oligoclonal, and changes in the growth and oncogenic potential of in vitro transformants occur during the derivation of the cell lines. Both of these features suggest that v-Abl expression must be complemented by changes in expression of one or more cellular genes for cells to acquire a fully malignant phenotype. Such genes could include other oncogenes or tumor suppressor genes. Among the latter is Tp53, a gene mutated in many spontaneous cancers. To determine if mutation of the Tp53 tumor suppressor gene plays a role in Abelson virus transformation, conformation-specific monoclonal antibodies were used to examine p53 expression in a panel of Abelson virus-transformed pre-B cells. Expression of mutant forms of p53 was detected in over 40% of the isolates. Sequence analysis revealed the presence of point mutations affecting the highly conserved central portion of the protein. These mutations interfered with the ability of p53 to activate transcription from a promoter containing p53-responsive elements and to induce apoptosis in response to DNA damage. In addition, cells expressing mutant forms of p53 induced a higher frequency of tumors with a more rapid course compared to transformants expressing wild-type p53. These data suggest that Tp53 is one important cellular gene involved in malignant transformation by Abelson virus.