Angelika K. Teresky

The Regulation of AMPK β1, TSC2, and PTEN Expression by p53: Stress, Cell and Tissue Specificity, and the Role of These Gene Products in Modulating the IGF-1-AKT-mTOR Pathways

The insulin-like growth factor 1 (IGF-1)-AKT-mTOR pathways sense the availability of nutrients and mitogens and respond by signaling for cell growth and division. The p53 pathway senses a variety of stress signals which will reduce the fidelity of cell growth and division, and responds by initiating cell cycle arrest, senescence, or apoptosis. This study explores four p53-regulated gene products, the beta1 and beta2 subunits of the AMPK, which are shown for the first time to be regulated by the p53 protein, TSC2, PTEN, and IGF-BP3, each of which negatively regulates the IGF-1-AKT-mTOR pathways after stress. These gene products are shown to be expressed under p53 control in a cell type and tissue-specific fashion with the TSC2 and PTEN proteins being coordinately regulated in those tissues that use insulin-dependent energy metabolism (skeletal muscle, heart, white fat, liver, and kidney). In addition, these genes are regulated by p53 in a stress signal-specific fashion. The mTOR pathway also communicates with the p53 pathway. After glucose starvation of mouse embryo fibroblasts, AMPK phosphorylates the p53 protein but does not activate any of the p53 responses. Upon glucose starvation of E1A-transformed mouse embryo fibroblasts, a p53-mediated apoptosis ensues. Thus, there is a great deal of communication between the p53 pathway and the IGF-1-AKT and mTOR pathways.

Declining p53 function in the aging process: a possible mechanism for the increased tumor incidence in older populations.

Cancer is a disease of aging. The accumulation of mutations in individual cells over a lifetime is thought to be the reason. In this work, we explored an additional hypothesis: could p53 function decline with age, which would contribute to an enhanced mutation frequency and tumorigenesis in the aging process? The efficiency of the p53 response to γ-irradiation was found to decline significantly in various tissues of aging mice from several inbred strains, including lower p53 transcriptional activity and p53-dependent apoptosis. This decline resulted from a decreased stabilization of the p53 protein after stress. The function of the Ataxia-telangiectasia mutated (ATM) kinase declined significantly with age, which may then be responsible for the decline of the p53 response to radiation. Declining p53 responses to other stresses were also observed in the cultured splenocytes from aging mice. Interestingly, the time of onset of this decreased p53 response correlated with the life span of mice; mice that live longer delay their onset of decreased p53 activity with time. These results suggest an enhanced fixation of mutations in older individuals because of the declining fidelity of p53-mediated apoptosis or senescence in response to stress, and they suggest a plausible explanation for the correlation between tumorigenesis and the aging process.

The gas5 gene is disrupted by a frameshift mutation within its longest open reading frame in several inbred mouse strains and maps to murine Chromosome 1

The gas5gene was identified in a screen for mRNAs induced in NIH3T3 fibroblasts following growth arrest (Schneider et al. 1988). gas5 message levels decline upon serum stimulation of quiescent mouse fibroblasts by what appears to be a posttranscriptional mechanism that may be dependent on protein kinase activity (Ciccarelli et al. 1990). gas5message levels may also be subject to transcriptional regulation that occurs in response to cellular differentiation (Coccia et al. 1992). Abundant levels ofgas5message have been observed in most adult mouse tissues examined, the exceptions being liver and spleen (Coccia et al.1992). Neural tube-specific expression of gas5 message during neural tube closure has been reported to occur in embryos of the SWV/Fnn mouse strain, but not in embryos of the LM/Bc/Fnn mouse strain. SWV/Fnn embryos are also more susceptible to hyperthermia-induced exencephaly than are LM/Bc/ Fnn embryos, which suggests a possible link between gas5 expression and susceptibility to neural tube defects (Vacha et al. 1997). Two predominantgas5messages are produced by alternative splicing, which eliminates exon 7 from one of the forms. There are only short open reading frames (ORFs) within the gas5message, the longest being either 123 or 117 base pairs depending on the splice variant (Coccia et al. 1992). Peptides encoded by these two ORFs, both with apparent molecular weights of about 8 kDa, have been produced in an in vitro translation system (Coccia et al. 1992); however, no evidence corroborating expression of these proteins in cells has been reported. Amplification of gas5cDNA by RT-PCR from the C57Bl/6J and 129Sv+++/J mouse strains resulted in a consistently smaller product from the 129 strain (data not shown). Sequence analysis revealed five fewer base pairs within exon 5 of the 129 derivedgas5cDNA owing to the absence of half of the 5 base pair tandem repeat TAGAATAGAA (Fig. 1A). Primers flanking this polymorphic region were designed to enable the analysis of genomic DNA. With these primers, tail DNA was assayed from an additional 11 inbred mouse strains and from outbred Swiss Webster andMus spretusmice (Fig. 1B). Of the 11 additional inbred strains tested, 5 produced PCR products of a size consistent with the 129 sequence. These strains were A/J, C3H/HeJ, LP/J, LT/SvA, and NZW/LacJ. The other 6 inbred strains produced PCR products of a size consistent with the C57Bl/6 sequence. These strains were AKR/J, BALB/cJ, DBA/2J, FVB/NJ, NOD/LtJ, and NON/ LtJ. The size of the PCR products from the outbred Swiss and spretusmice were both consistent with the C57Bl/6 sequence. Since the intact tandem repeat was found in the species Mus spretus, this seems more likely to represent the wild-type sequence. However, the high proportion of inbred mouse strains harboring the 5-bp deletion suggests either that this sequence has been remarkably unstable within the inbred strains or that this polymorphism was present within and inherited from the founder population. Three markers, D12Mit46, D12Mit64,and D12Mit54, which should flank thegas5gene based upon its previously reported map position on mouse Chromosome (Chr) 12 (Dietrich et al. 1996), failed to show evidence of linkage on a panel of 42 C57B16×129 Correspondence to: A.J. Levine Fig. 1. (A) Nucleotide sequence of the 5-bp polymorphism identified within exon 5 of thegas5 gene. Capital letters indicate the nucleotide sequence of exon 5 of the gas5 gene, while lower-case letters indicate flanking intron/exon boundary sequences (Coccia et al. 1992). Asterisks (*) denote the 5-bp that are absent in the 129 strain. The top and bottom arrows del ineate the sequences of the forward primer 5 8TAACTATTTGTTTGTTGTAGGTGC-38 and of the reverse primer 5 8TGACTTTACCATTTCATTTTCTGG-38 used for PCR amplification from genomic DNA.(B) Genomic PCR analysis of exon 5 of the gas5gene from 13 different inbred mouse strains 129Sv+ ++/J, C57Bl/6J, A/J, AKR/J, BALB/cJ, C3H/HeJ, DBA/2J, FVB/NJ, LP/J, LT/Sv-A, NOD/ LtJ, NON/LtJ, NZW/LacJ and from outbred Swiss Webster and Mus spretus mice. The PCR products of either 55 or 60 bp were resolved on 15% acrylamide gels and detected by ethidium bromide staining. Mammalian Genome 9, 773–774 (1998).

Teratocarcinoma transplantation rejection loci: AnH-2-linked tumor rejection locus

Embryoid bodies (ascites tumor) from a 129/Sv transplantable teratocarcinoma produce tumors (100%) in syngenic 129/Sv mice but fail to form tumors (3–6%) in BALB/c mice, C3H/He mice and C57BL/6 mice, in spite of the fact that the malignant stem cells of this tumor do not express detectable H-2 antigens. The available evidence indicates that this allogeneic tumor restriction has an immunological basis; 100% of the F1 hybrid mice between 129/Sv and the three other inbred mouse strains accept the 129/Sv teratocarcinoma. The backcross and F2 mice segregate the BALB/c, C3H/He and C57BL/6 tumor transplantation rejection loci in a manner that indicates that each of these inbred strains of mice contain one to two major transplantation rejection loci. A linkage analysis in the BALB/c and C3H/He backcross and F2 generations indicates that these mice have a teratocarcinoma transplantation rejection locus on chromosome 17, about eight to nine recombination units from theH- 2 complex. An F1 complementation analysis between allogeneic mice that each reject teratocarcinomas tumors (BALB/c × C57BL/6 and C3H/He × C57BL/6), indicates that the C57BL/6 mice have the 129/Sv tumor-accepting (sensitive) allele at theH-2-linked locus but reject teratocarcinomas because of antigenic differences at a second locus.While these major teratocarcinoma transplantation rejection loci determine the acceptance or rejection of a tumor by a mouse injected with high doses of tumor tissue (750 μg of tumor protein), evidence is presented for a number of minor genetic factors that can (1) affect the efficiency of tumor rejection and (2) cause complete tumor rejection at lower tumor doses (7.5–75 μg of tumor protein).

Permanent teratocarcinoma-derived cell lines stabilized by transformation with SV40 and SV40tsA mutant viruses.

Publisher Summary This chapter discusses the permanent teratocarcinoma-derived cell lines stabilized by transformation with SV40 and SV40tsA mutant viruses. Mouse teratocarcinoma cells derived from embryoid bodies of 129SvS1 mice were cultured in vitro to permit their differentiation. These cells were then infected with three different SV40tsA mutants at 32°C, and six cloned cell lines (SVtsA-teratocarcinoma derived cells) were derived from these cultures. All six cell lines expressed the SV40 tumor antigen (94,000 MW) in a temperature-sensitive fashion, with lower levels of this antigen detected at the nonpermissive temperature. Mock infected teratocarcinoma-derived cells did not give rise to permanent cell lines demonstrating the necessity of the virus or its products in the formation of the teratocarcinoma-derived cell lines. Temperature shift experiments demonstrated that the viral A gene product plays an essential role in the ability of the SVtsA cell lines to form colonies on plastic surfaces in 2% serum-containing medium, on top of monolayer cell cultures in 10% serum-containing medium, or in agar suspension cultures.

Genetic mapping of the embryonal carcinoma transplantation resistance locus Gt(B6) to mouse Chromosome 8.

Abstract Cytotoxic T lymphocytes play a predominant role in allograft rejection. They mediate this process through recognition of foreign major histocompatibility complex (MHC) class I surface molecules encoded at the H2 locus. Embryonal carcinoma cells, the undifferentiated, neoplastic derivatives of primordial germ cells, typically lack detectable MHC class I gene expression. Despite this, embryonal carcinoma cells are subject to allograft rejection in several different mouse strains. In many instances, the H2 locus appears to be genetically linked to resistance. However, rejection of allografts of the F9 embryonal carcinoma cell line, a nullipotent cell line derived from the 129 mouse strain, does not appear to be H2-linked. Resistance to F9 tumor formation in the C57BL/6 mouse strain has been attributed to a single, unidentified locus termed Gt(B6). To genetically map the Gt(B6) locus, a total of 463 (C57BL/6×129)F2 mice were challenged with F9 cells, and 78 tumor-resistant mice were identified. Markers encompassing two candidate regions, the H2 locus on Chromosome (Chr) 17 and a second candidate locus on Chr 2, showed no indication of linkage to the resistance phenotype. Instead, results of a genome wide scan implicated mouse Chr 8, and evidence is presented demonstrating that the Gt(B6) locus maps to a region of less than 10 cM on the medial portion of Chr 8.