Molly Kulesz-Martin

Endogenous p53 protein generated from wild-type alternatively spliced p53 RNA in mouse epidermal cells

We previously demonstrated that a wild-type alternatively spliced p53 (p53as) RNA exists in mouse cultured cells and normal mouse tissues at approximately 25 to 33% of the level of the major p53 RNA form. The alternative RNA transcript is 96 nucleotides longer than the major transcript as a result of alternative splicing of intron 10 sequences. The protein expected to be generated from the p53as transcript is 9 amino acids shorter than the major p53 protein and has 17 different amino acids at the carboxyl terminus. We report here that p53as protein exists in nontransformed and malignant epidermal cells and is localized to the nucleus. In addition, p53as protein is preferentially expressed during the G2 phase of the cell cycle and in cells with greater than G2 DNA content compared with the major p53 protein, which is preferentially expressed in G1. The p53as immunoreactivity is elevated and shifted to the G1 phase of the cell cycle following actinomycin D treatment of nontransformed cells but not malignant cells. In view of the dimerization and tetramerization of p53 protein which may be necessary for its DNA binding and transcriptional activation activities, the presence of p53as protein in cells has important implications for understanding the physiological function(s) of the p53 gene.

Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells.

A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence‐specific DNA‐binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero‐oligomers when co‐translated in vitro, resulting in inactivation of p53as DNA‐binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero‐oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild‐type p53 gene.

Sliding into home: facilitated p53 search for targets by the basic DNA binding domain.

p53 is a unique DNA binding protein with two distinct DNA binding domains, the evolutionarily conserved central domain and the C-terminal basic DNA binding domain (BD domain). The presence of two separate DNA binding domains with distinct DNA binding properties distinguishes p53 from other DNA binding proteins. Transcription factors generally bind DNA with sequence specificity but not with DNA structure specificity. DNA repair proteins generally bind DNA with structure specificity without sequence specificity. Even in the p53 gene family, the BD domain is a unique feature of vertebrate p53 proteins that is absent in p63, p73 and p53 homologues in primitive species like squid, Drosophila and Caenorhabdities. elegans. A central question is why p53 requires two DNA binding domains for its role as a tumor suppressor.

Analysis of DNA damage at the dinucleoside monophosphate level: Application to the formamido lesion

The dinucleoside monophosphates d(TpG), d(TpC), and d(TpT) were X-irradiated in oxygenated solution. In each case the modification of the dinucleoside in which the thymine base is degraded to a formamido remnant was observed as a principal product. The hydrolysis of the phosphoester bond of formamido-modified dinucleosides is much slower than that of the corresponding unmodified dinucleosides. This effect is also observable in the hydrolysis of irradiated DNA, where hydrolysis by nuclease P1 (plus acid phosphatase) generates the modified dinucleosides d(TFpN), TF being the modified thymidine. The total yield of the formamido lesion in all its forms, d(TFpN), exceeds the yield of any other base modification.

Steady-State Cultures of Human Skin

Pretransplantation cultivation of adult human skin has been optimized for rapid and prolonged outgrowth of epidermal cells from tissue explants using autonomic-perfusion, thin-layer culture technology (steady-state). This system fostered growth of autologous mesenchymal elements via critical control of the culture environment. The resulting cellular outgrowth maintained a balanced epithelial-dermal relationship, contained keratinocytes as well as minority epidermal cells, melanocytes and possibly Langerhans cells. Critical control of culture pH and osmolarity was found to enhance epithelial cell proliferation.

Molecular Markers for Colon Cancer: Where Do We Go from Here?

Molecular and biochemical markers are greatly needed for cancer diagnosis and prognosis and for the selection and monitoring of treatments, yet they remain a major challenge. The report of Nemunaitis et al. in this issue analyzes patient survival and mutations of the K-rus oncogene and the p.53 tumor suppressor gene, and their protein expression levels by immunohistochemistry, in 27 colon cancer patients who failed 5-FU therapy and were treated with the matrix metalloproteinase inhibitor Marimastat (BB25 16). The results indicated that K-ras mutation but not expression was associated with decreased survival and that neither pS3 mutation nor expression independently predicted survival, although the combination of mutations of both K-rus and pS3 were associated with the least survival. Measurement of molecular markers is not standard practice in the diagnosis of colon cancer but is being carried out in a fraction (totaling thousands) of cases in the National Surgical Adjuvant Breast and Bowel Project (NSABP) clinical trials to point the way for their future use in individualized tumor typing and optimized treatment. (An example of collaborating NSABP investigators is given in ref. 1 .) K-rus and p53 are likely candidates for molecular diagnosis because defects in each occur at high frequency during human colon carcinogenesis. It is clear that mutation of K-rus leads to overexpression and activation of its oncogenic potential, whereas mutation of pS3 often results in overexpression of a functionally defective protein, interfering with its modulation of cell growth, apoptosis, or repair and even contributing to genomic instability and tumor progression. The current results show a need for further analysis of these prognostic indicators but may not necessarily allow one to conclude that survival in colon cancer patients generally is predicted better by mutations in K-rcts than by p53 genotype and immunohistochemistry. The results of Nemunaitis et al. conflict with those of Pricolo et al. (21), who reported that K-ras mutations alone were not conclusively associated with poorer long-term prognosis in patients with colorectal cancer, and with those of Pricolo et al. (3) and Auvinen et al. (4), who found that p53 mutation or expression was negatively associated with survival. The differences could be due to sample size and characteristics and methods of detection. Sample size was smaller in the current study (27 cases) than in these other studies (70, 141, and 144 cases, respectively). None of the cases in the other studies were subjected to adjuvant chemotherapy, and the percentages of pS3 mutations were higher (61 and 66%) compared with the current study sample (7/27 mutant and 13/27 immunohistochemistry positive). Differences could be due to the small sample size or to bias in the sampling, introduced, for example, by selection of tumors by 5-FU treatment. Another consideration in selection of patient populations is exclusion of hereditary non-polyposis colorectal

Cultured Human Skin: Heterotransplantation

We have reported that human skin, cultured in a controlled environment system (steady state), yields epidermis-like growth from adult donor split thickness specimens. A procedure for transplantation of such cellular outgrowths to athymic nude mice for functional and morphological evaluations is reported. Successful transplants were definitely scored by the presence of histologic epidermal markers and human glucose phosphate isomerase. We suggest that the transplantation procedures reported here may contribute to the knowledge required for the use of autologous cultured skin in patients with large skin wounds.