Loss of Heterozygosity at theInk4a/ArfLocus Facilitates Abelson Virus Transformation of Pre-B Cells

Abstract

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.Abelson murine leukemia virus (Ab-MLV) is a rapidly transforming retrovirus that can induce lymphomas in vivo and transform pre-B cells and immortalized fibroblast cell lines in vitro (reviewed in references 39 and 40). The virus carries the v-abl oncogene, and the protein tyrosine kinase it encodes is required for transformation. Despite the strong growth-stimulatory signal provided by the v-Abl protein, Ab-MLV-induced transformation is a multistep process both in vivo and in vitro (16,17,59,60), suggesting that multiple cellular changes are required before a cell becomes fully malignant. Analysis of primary pre-B-cell transformants in vitro has revealed that the p53 tumor suppressor gene and the p19Arf gene, the product of which regulates p53 function (reviewed in references 35 and 46), are intimately involved in the process by which these cells evolve to become fully malignant established cell lines (37,52,54). About 50% of all transformants contain mutations affecting p53 (52), and many others express very low levels of the p19Arf protein, a molecule that stabilizes p53, thereby enhancing its function (37).The factors involved in tumor progression in the Ab-MLV system have received limited attention, and most studies using Ab-MLV or other oncogenic retroviruses have focused on oncogene cooperativity (reviewed in reference 42). However, in many other types of tumors, dominant growth-stimulatory signals generated by oncogenes cooperate with the loss of growthsuppressive signals provided by tumor suppressor genes (26,45). Alterations in the p53 and NF1 tumor suppressor genes have been reported in some retrovirus-induced tumors (3-6, 53, 56, 61). Other studies have identified chromosomal regions ...

The Absence of Msh2 Alters Abelson Virus Pre-B-Cell Transformation by Influencing p53 Mutation

“…The inherently high mutation frequency found in the absence of Msh2 (1,12,44) likely increases the chances that a mutation facilitating escape from crisis will occur. Although loss of p19Arf expression is another mechanism by which primary transformants recover from crisis (41), mutations affecting the p19Arf coding sequence have not been observed in any Ab-MLV transformants, including those studied here (35,54). The mechanism by which p19Arf is regulated in Ab-MLV transformants has not been elucidated.…”

Rapid, Stabilizing Palindrome Rearrangements in Somatic Cells by the Center-Break Mechanism

“…Permanent cell lines were therefore established by infection of Line 78 fetal livers with Ab-MLV, a procedure that gives rise to pre-B-cell-like transformants (29). Ab-MLV-transformed lymphoid cell lines were chosen specifically because they are stably blocked in differentiation, have a consistent population doubling time, are simple to culture without added growth factors, and typically exhibit stable, diploid chromosome counts (27,31,34,35). A concern with this system is that palindrome modification or hairpin nicking in Ab-MLV transformants might exhibit significant pre-B-cell-specific attributes; however, investigations with transiently transfected palindromes and hairpins previously failed to uncover any major differences between lymphoid and nonlymphoid cells (20,21).…”

The biologic properties of leukemias arising from BCR/ABL-mediated transformation vary as a function of developmental origin and activity of the p19ARF gene

“…In vitro, transformation of precursor B cells by v-abl was shown to be greatly facilitated by null mutations at the p16 INK4A /p19 ARF locus; further studies associated this activity specifically with loss-of-function mutations involving the p19 ARF gene. [18][19][20] The aforementioned studies suggest that the gene products of this tumor suppressor locus, especially p19 ARF , might be crucial for normal lymphocyte development. Indeed, the p16 INK4A /p19 ARF -null (exons 2 and 3) and p19 ARF -null (exon 1␤) mice develop aggressive lymphoproliferative diseases, among many other types of tumors seen in the adult mice.…”