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T Jacks

Publications and source records attributed to T Jacks.

124 records · Page 7Linked to original sources
PubMed1993

p53-dependent apoptosis modulates the cytotoxicity of anticancer agents.

Although the primary cellular targets of many anticancer agents have been identified, less is known about the processes leading to the selective cell death of cancer cells or the molecular basis of drug resistance. p53-deficient mouse embryonic fibroblasts were used to examine systematically the requirement for p53 in cellular sensitivity and resistance to a diverse group of anticancer agents. These results demonstrate that an oncogene, specifically the adenovirus E1A gene, can sensitize fibroblasts to apoptosis induced by ionizing radiation, 5-fluorouracil, etoposide, and adriamycin. Furthermore, the p53 tumor suppressor is required for efficient execution of the death program. These data reinforce the notion that the cytotoxic action of many anticancer agents involves processes subsequent to the interaction between drug and cellular target and indicate that divergent stimuli can activate a common cell death program. Consequently, the involvement of p53 in the apoptotic response suggests a mechanism whereby tumor cells can acquire cross-resistance to anticancer agents.

Adenovirus E1A Proteins↗
PubMed1993

Generation of normal lymphocyte populations by Rb-deficient embryonic stem cells.

BACKGROUND: Mice homozygous for a loss-of-function mutation of the recombination-activating gene-2 (RAG 2), which is required for the rearrangement of antigen receptor genes, do not produce mature B and T lymphocytes. But chimeric mice that result from injection of normal embryonic stem (ES) cells into blastocysts from RAG2-deficient mice develop normal mature lymphocyte populations, all of which are derived from the injected ES cells; we have called this process RAG2-deficient blastocyst complementation. Using ES cells with homozygous mutations, RAG-2-deficient blastocyst complementation could provide a physiological assay with which to determine the potential role of almost any gene in the development and/or function of lymphocytes. To test the general utility of this system, we have used it to test the differentiation-potential of ES cells that harbor homozygous loss-of function mutations of their retinoblastoma susceptibility (Rb) gene loci. We chose Rb for this analysis because of its widespread function in the control of the cell cycle and cell differentiation, the adverse effect of homozygous germline mutations of Rb on hematopoiesis in fetal liver, and the embryonic lethality that results when the homozygous Rb mutation is introduced into the germline. RESULTS: Homozygous Rb mutant ES cells can develop into phenotypically normal, mature B and T lymphocytes in the RAG-2-deficient background. Strikingly, Rb-deficient B and T cells do not have major defects in either activation or function. CONCLUSION: We have demonstrated the efficacy of the RAG-2-deficient blastocyst complementation system for evaluating the role of critical genes in lymphocyte development. Our results indicate that Rb expression is not intrinsically required for B-cell or T-cell function, despite the normally high levels of Rb expressed in lymphoid cells.

Journal Article↗
PubMed1993

A nonpeptidyl growth hormone secretagogue.

A nonpeptidyl secretagogue for growth hormone of the structure 3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2'-(1H-tetrazol-5 -yl) (1,1'-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamid e (L-692,429) has been identified. L-692,429 synergizes with the natural growth hormone secretagogue growth hormone-releasing hormone and acts through an alternative signal transduction pathway. The mechanism of action of L-692,429 and studies with peptidyl and nonpeptidyl antagonists suggest that this molecule is a mimic of the growth hormone-releasing hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6). L-692,429 is an example of a nonpeptidyl specific secretagogue for growth hormone.

Amino Acid Sequence↗
PubMed1993

p53 is required for radiation-induced apoptosis in mouse thymocytes.

The p53 tumour suppressor gene is the most widely mutated gene in human tumorigenesis. p53 encodes a transcriptional activator whose targets may include genes that regulate genomic stability, the cellular response to DNA damage, and cell-cycle progression. Introduction of wild-type p53 into cell lines that have lost endogenous p53 function can cause growth arrest or induce a process of cell death known as apoptosis. During normal development, self-reactive thymocytes undergo negative selection by apoptosis, which can also be induced in immature thymocytes by other stimuli, including exposure to glucocorticoids and ionizing radiation. Although normal negative selection involves signalling through the T-cell receptor, the induction of apoptosis by other stimuli is poorly understood. We have investigated the requirement for p53 during apoptosis in mouse thymocytes. We report here that immature thymocytes lacking p53 die normally when exposed to compounds that may mimic T-cell receptor engagement and to glucocorticoids but are resistant to the lethal effects of ionizing radiation. These results demonstrate that p53 is required for radiation-induced cell death in the thymus but is not necessary for all forms of apoptosis.

Animals↗
PubMed1993

Neurofibromatosis type 1 gene product (neurofibromin) associates with microtubules.

The neurofibromatosis type 1 (NF1) gene was recently identified by positional cloning and found to encode a protein with structural and functional homology to mammalian and yeast GTPase-activating proteins (GAPs). Using antibodies directed against the NF1 gene product, a protein of approximately 250 kDa was identified and termed neurofibromin. Double-indirect immunofluorescent labeling with anti-neurofibromin and anti-tubulin antibodies demonstrates that neurofibromin associates with cytoplasmic microtubules. Immunoblotting of microtubule-enriched cytoplasmic fractions, using antibodies generated against neurofibromin, shows that neurofibromin copurifies with microtubules. When portions of neurofibromin are expressed in Sf9 insect cells they associate with polymerized microtubules; furthermore, the critical residues for this interaction reside within the GAP-related domain of neurofibromin. The unexpected association of neurofibromin with microtubules suggests that neurofibromin is involved in microtubule-mediated intracellular signal transduction pathways.

3T3 Cells↗
PubMed1993

Cell cycle-specific association of E2F with the p130 E1A-binding protein.

Association of the E2F transcription factor with the pRb and p107 proteins appears to regulate the activity of E2F and, in turn, affect cell cycle progression. We found, however, that pRb and p107 are only minor E2F-associated proteins in G0/G1 mouse fibroblasts, and we sought to identify the major E2F partner protein in these cells. Because the adenovirus E1A oncoprotein seemed able to bind to the G0 E2F partner, we enriched for proteins that associated both with an E2F-binding site DNA column and with E1A. The major species in G0 and early G1 fibroblasts detected with this approach had properties identical to the pRb- and p107-related p130 protein. In serum-stimulated cells, p107 replaced p130 as the major E2F-associated protein near the G1/S border, concomitant with an increase in p107 protein levels. p130-E2F complexes resembled p107-E2F complexes in their ability to bind to cyclin-cdk kinases, and they appeared to be associated with the cyclin E-cdk2 kinase in late G1 cells. These observations indicate that E2F transcription factors are regulated by a succession of partner proteins with which they associate during defined stages of the cell cycle.

3T3 Cells↗
PubMed1993

Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival.

Oct-2, a POU homeo domain transcription factor, is believed to stimulate B-cell-restricted expression of immunoglobulin genes through binding sites in immunoglobulin gene promoters and enhancers. To determine whether Oct-2 is required for B-cell development or function, or has other developmental roles, the gene was disrupted by homologous recombination. Oct-2-l- mice develop normally but die within hours of birth for undetermined reasons. Mutants contain normal numbers of B-cell precursors but are somewhat deficient in IgM+ B cells. These B cells have a marked defect in their capacity to secrete immunoglobulin upon mitogenic stimulation in vitro. Thus, Oct-2 is not required for the generation of immunoglobulin-bearing B cells but is crucial for their maturation to immunoglobulin-secreting cells and for another undetermined organismal function.

Animals↗
PubMed1992

A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.

Cell cycle checkpoints can enhance cell survival and limit mutagenic events following DNA damage. Primary murine fibroblasts became deficient in a G1 checkpoint activated by ionizing radiation (IR) when both wild-type p53 alleles were disrupted. In addition, cells from patients with the radiosensitive, cancer-prone disease ataxia-telangiectasia (AT) lacked the IR-induced increase in p53 protein levels seen in normal cells. Finally, IR induction of the human GADD45 gene, an induction that is also defective in AT cells, was dependent on wild-type p53 function. Wild-type but not mutant p53 bound strongly to a conserved element in the GADD45 gene, and a p53-containing nuclear factor, which bound this element, was detected in extracts from irradiated cells. Thus, we identified three participants (AT gene(s), p53, and GADD45) in a signal transduction pathway that controls cell cycle arrest following DNA damage; abnormalities in this pathway probably contribute to tumor development.

Amino Acid Sequence↗
PubMed1992

Effects of an Rb mutation in the mouse.

The retinoblastoma gene is mutated in several types of human cancer and is the best characterized of the tumour-suppressor genes. A mouse strain has been constructed in which one allele of Rb is disrupted. These heterozygous animals are not predisposed to retinoblastoma, but some display pituitary tumours arising from cells in which the wild-type Rb allele is absent. Embryos homozygous for the mutation die between days 14 and 15 of gestation, exhibiting neuronal cell death and defective erythropoiesis.

Amino Acid Sequence↗
PubMed1992

Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53.

Gene amplification occurs at high frequency in transformed cells (10(-3)-10(-5)), but is undetectable in normal diploid fibroblasts (less than 10(-9)). This study examines whether alterations of one or both p53 alleles were sufficient to allow gene amplification to occur. Cells retaining one wild-type p53 allele mimicked the behavior of primary diploid cells: they arrested growth in the presence of drug and failed to demonstrate amplification. Cells losing the second p53 allele failed to arrest when placed in drug and displayed the ability to amplify at a high frequency. Thus, loss of wild-type p53 may lead to amplification, possibly caused by changes in cell cycle progression. Other determinants can by-pass this p53 function, however, since tumor cells with wild-type p53 have the ability to amplify genes.

Animals↗
PubMed1988

Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region.

The gag-pol protein of Rous sarcoma virus (RSV), the precursor to the enzymes responsible for reverse transcription and integration, is expressed from two genes that lie in different translational reading frames by ribosomal frameshifting. Here, we localize the site of frameshifting and show that the frameshifting reaction is mediated by slippage of two adjacent tRNAs by a single nucleotide in the 5' direction. The gag terminator, which immediately follows the frameshift site, is not required for frameshifting. Other suspected retroviral frameshift sites mediate frameshifting when placed at the end of RSV gag. Mutations in RSV pol also affect synthesis of the gag-pol protein in vitro. The effects of these mutations best correlate with the potential to form an RNA stem-loop structure adjacent to the frameshift site. A short sequence of RSV RNA, 147 nucleotides in length, containing the frameshift site and stem-loop structure, is sufficient to direct frameshifting in a novel genetic context.

Amino Acid Sequence↗
PubMed1988

Characterization of ribosomal frameshifting in HIV-1 gag-pol expression.

Based on precedents from other retroviruses, the precursor of the human immunodeficiency virus (HIV-1) reverse transcriptase is predicted to be a polyprotein with a relative molecular mass (Mr) of 160,000 (160K) encoded by both the viral pol gene and the upstream gag gene. These two genes lie in different translational reading frames, with the 3' end of gag overlapping the 5' end of pol by 205 or 241 nucleotides. Thus, production of the gag-pol fusion protein would require either messenger RNA processing or translational frameshifting. The latter mechanism has been shown in the synthesis of the gag-pol proteins of two other retroviruses, Rous sarcoma virus (RSV) and mouse mammary tumour virus (MMTV). Here we report that translation of HIV-1 RNA synthesized in vitro by SP6 RNA polymerase yields significant amounts of a gag-pol fusion protein, indicating that efficient ribosomal frameshifting also occurs within the HIV-1 gag-pol overlap region. Site-directed mutagenesis and amino-acid sequencing localized the site of frameshifting to a UUA leucine codon near the 5' end of the overlap.

Amino Acid Sequence↗
PubMed1987

Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins.

The primary translation products of retroviral pol genes are polyproteins initiated in an upstream gene (gag). To investigate the manner in which the gag-initiated polyproteins of the mouse mammary tumor virus are produced, we determined the nucleotide sequence of a 1.8-kilobase DNA fragment that spans the region between gag and pol in the C3H strain of mouse mammary tumor virus. The sequence reveals three overlapping open reading frames: the first encodes products of gag (p27gag and p14gag); the second encodes a protein domain of unknown function (termed X) that is highly related to a similarly positioned sequence in simian type D retroviruses and the viral protease (pro); and the third encodes the reverse transcriptase. The reading frames are organized to permit uninterrupted readthrough from gag to pol if ribosomal frameshifts occur in the -1 direction within each of the two overlapping regions, one of which is 16 nucleotides in length and the other 13 nucleotides. Cell-free translation of RNA containing these overlap regions shows that fusion of the reading frames by ribosomal frameshifting occurs efficiently: about one-fourth of the ribosomes traversing the gag-X/pro overlap and one-tenth traversing the X/pro-pol overlap shift frames, generating gag-related polyproteins in ratios similar to those observed in vivo. Synthetic oligonucleotides containing either of the overlap regions inserted into novel contexts do not induce frameshifting; hence the overlapping portions of the reading frames are not sufficient to induce a frameshift event, and a larger sequence context or secondary structure may be implicated.

Amino Acid Sequence↗
PubMed1985

Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting.

The pol gene of Rous sarcoma virus is positioned downstream of the gag gene in a different, briefly overlapping reading frame; nevertheless, the primary translation product of pol is a gag-pol fusion protein. Two mechanisms, ribosomal frameshifting and RNA splicing, have been considered to explain this phenomenon. The frameshifting model is supported by synthesis of both gag protein and gag-pol fusion protein in a cell-free mammalian translation system programmed by a single RNA species that was synthesized from cloned viral DNA with a bacteriophage RNA polymerase. Under these conditions, the ratio of the gag protein to the fusion protein (about 20 to 1) is similar to that previously observed in infected cells, the frameshifting is specific for the gag-pol junction, and it is unaffected by large deletions in gag. In addition, synthesis of the fusion protein is ten times less efficient in an Escherichia coli cell-free translation system and cannot be explained by transcriptional errors or in vitro modification of the RNA. Ribosomal frameshifting may affect production of other proteins in higher eukaryotes, including proteins encoded by several retroviruses and transposable elements.

Animals↗
PubMedDate not supplied

Sunburn and p53 in the onset of skin cancer.

Squamous cell carcinoma of the skin (SCC) can progress by stages: sun-damaged epidermis, with individual disordered keratinocytes; actinic keratosis (AK), spontaneously regressing keratinized patches having aberrant cell differentiation and proliferation; carcinoma in situ; SCC and metastasis. To understand how sunlight acts as a carcinogen, we determined the stage at which sunlight mutates the p53 tumour-suppressor gene and identified a function for p53 in skin. The p53 mutations induced by ultraviolet radiation and found in > 90% of human SCCs were present in AKs. Inactivating p53 in mouse skin reduced the appearance of sunburn cells, apoptotic keratinocytes generated by overexposure to ultraviolet. Skin thus appears to possess a p53-dependent 'guardian-of-the-tissue' response to DNA damage which aborts precancerous cells. If this response is reduced in a single cell by a prior p53 mutation, sunburn can select for clonal expansion of the p53-mutated cell into the AK. Sunlight can act twice: as tumour initiator and tumour promoter.

Animals↗