p16CDKN2 specifically binds to and inhibits the cyclin-dependent kinases CDK4 and CDK6, which function as regulators of cell cycle progression in G 1 by contributing to the phosphorylation of the retinoblastoma protein (pRB). Human cell lines lacking functional pRB contain high levels of p16 RNA and protein, suggesting a negative feedback loop by which pRB might regulate p16 expression in late G 1 . By a combination of nuclear run-on assays and promoter analyses in human fibroblasts expressing a temperature-sensitive simian virus 40 T antigen, we show that p16 transcription is affected by the status of pRB and define a region in the p16 promoter that is required for this response. However, the effect is not sufficient to account for the differences in p16 RNA levels between pRB-positive and -negative cells. Moreover, p16 RNA is extremely stable, and the levels do not change appreciably during the cell cycle. Primary human fibroblasts express very low levels of p16, but the RNA and protein accumulate in late-passage, senescent cells. The apparent overexpression of p16 in pRB-negative cell lines is therefore caused by at least two factors: loss of repression by pRB and an increase in the number of population doublings.CDKN2, also referred to as INK4A, CDK4I, and MTS1 (23,36,49), is a putative tumor suppressor gene on human chromosome 9p21 that is genetically linked with familial inheritance of malignant melanoma and is inactivated by a variety of mechanisms in a broad spectrum of human cancers (reviewed in references 22 and 51). CDKN2 encodes a 156-amino-acid protein, designated p16, that specifically binds to and inactivates the cyclin-dependent kinases (CDKs) CDK4 and CDK6 (16,19,41,49). These kinases are the major catalytic partners for cyclins D1, D2, and D3 and collaborate with cyclin E-CDK2 in controlling the G 1 /S transition in mammalian cells (reviewed in references 9, 43, and 53).One of the critical substrates of the G 1 -specific cyclin-dependent kinases is the product of the retinoblastoma gene (pRB) which, in its hypophosphorylated state, exerts a negative influence on G 1 progression (42,63). Since phosphorylation of pRB equates with its functional inactivation, a relatively robust model has emerged in which the role of the cyclin D-dependent kinases is to initiate the phosphorylation of pRB (9,52,63). Consistent with such a scheme, antibody neutralization studies have indicated that D-type cyclin function is required up to a point in G 1 coincident with the block imposed by pRB (3,44). This requirement is lost in cells lacking functional pRB (29,30). Ectopic expression of p16, which specifically interferes with the cyclin D-dependent kinases CDK4 and CDK6, imposes an analogous G 1 block that is also dependent on functional pRB (14,24,31,34,48). Conversely, ectopic expression of D cyclins can accelerate G 1 progression, particularly when coupled with elevated expression of cyclin E (2,20,35,(44)(45)(46). The model can readily explain the role of p16 as a tumor suppressor, since loss of function will...

No abstract

No abstract

No abstract

No abstract

We sequenced two recombinant DNA clones constituting a single provirus of the milk-transmitted mouse mammary tumor virus characteristic of BR6 mice. The complete provirus is 9,901 base pairs long, flanked by 6 base-pair duplications of cellular DNA at the site of integration. Five extensive blocks of open reading frame corresponding to the gag gene, the presumed protease, the pol and env genes, and the open reading frame orf within the long terminal repeat of the provirus were readily discernible. Translation of gag, protease, and pol involved three different translational reading frames to produce the three overlapping polyprotein precursors Pr77, Pr110, and Prl60 found in virus-infected cells. Synthesis of the reverse transcriptase and endonuclease therefore required two separate frameshifts to suppress the termination codons at the ends of the Pr77 and PrllO domains. Direct evidence is presented for translational readthrough of both stop codons in an in vitro protein synthesis system.

Mouse embryonal carcinoma cell lines that differ in their patterns of expression of the potential oncogene int‐2 have been exploited in a structural analysis of the multiple RNA transcripts characteristic of this gene. Ribonuclease protection experiments indicate that four major classes of int‐2 RNA initiate at heterogeneous cap sites within two distinct promoter regions, P1 and P2, spanning approximately 50 and 150 bp respectively. The more downstream promoter P2 is located in a region of the DNA that constitutes an intron in RNA transcripts that initiate at the upstream promoter P1. Otherwise, all four RNA structures share the same splice donor and acceptor sites that define the boundaries of the second and third exons. Further complexity arises through usage of two distinct polyadenylation signals, both variants of the normal consensus, that are separated by 1100 bp. Despite these structural variations, the results suggest that all four major classes of RNA encode the same protein product which shows significant homology to the family of heparin‐binding proteins typified by basic fibroblast growth factor (FGF).

BiochemistryThe mouse homolog of the hst/k-FGF gene is adjacent to int-2 and is activated by proviral insertion in some virally induced mammary tumors ( ABSTRACTThe fibroblast growth factor-related protooncogenes, int-2 and hst/k-FGF, are within 17 kilobase pairs of one another on mouse chromosome 7 and are in the same transcriptional orientation. Approximately 70% of tumors induced in BR6 mice by mouse mammary tumor virus have proviral insertions adjacent to the int-2 gene. We find that the murine homolog of the hst/k-FGF gene can also be transcriptionally activated by the insertion of mouse mammary tumor virus DNA either upstream or downstream of the gene. In most tumors, only one of these adjacent genes is activated, but in some cases both genes are expressed. One of the hst-expressing tumors also has a virally activated int-3 gene. At least five distinct cellular genes (int-i, -2, -3, 4, and hst/k-FGF) can therefore contribute, either singly or in concert, to the development of histologically indistinguishable mammary tumors in mice infected by mouse mammary tumor virus.