Cellular adhesion regulates p53 protein levels in primary human keratinocytes.

To gain insight into p53 tissue-specific regulatory pathways and biological activities, we investigated mechanisms that may account for the elevated levels of p53 protein in human foreskin keratinocytes, relative to levels in dermal fibroblasts in vitro. Here, we report that the loss of cell anchorage resulted in an approximately 5-fold decrease in p53 levels in keratinocytes, which was reversible upon reattachment of cells to a substratum. In contrast, fibroblasts did not exhibit such adhesion-dependent regulation of p53 protein. Furthermore, p53 function was attenuated in keratinocytes relative to fibroblasts. These results link p53 to cell adhesion pathways and may provide a molecular basis for epigenetic differences in the maintenance of genomic stability among normal cell types.

Human mammary epithelial cells exhibit a differential p53-mediated response following exposure to ionizing radiation or UV light.

The tumor suppressor protein, p53, plays a critical role as a transcriptional activator of downstream target genes involved in the cellular response to DNA damaging agents. We examined the cell cycle checkpoint response of human mammary epithelial cells (HMEC) and their isogenic fibroblast counterparts to ionizing (IR) and ultraviolet (UV) radiation, two genotoxic agents whose DNA damage response pathways involve p53. Using flow cytometric analysis, we found that both mortal and immortalized HMEC, which contain wild-type p53 sequence, do not exhibit a G1 arrest in response to IR, but show an intact G2 checkpoint. Supportive evidence from Western analyses revealed that there was neither an increase in p53 nor one of its downstream targets, p21WAF1, in HMEC exposed to IR. In contrast, isogenic mammary fibroblasts arrest at the G1 checkpoint and induce the p53 and p21WAF1 proteins following IR. By comparison, HMEC exposed to UV displayed an S phase arrest and induced the expression of p53 and p21WAF1. Our results show that the cellular response to DNA damage depends on both the type of damage introduced into the DNA and the specific cell type.

srd1, a Saccharomyces cerevisiae suppressor of the temperature-sensitive pre-rRNA processing defect of rrp1-1.

We define a new gene, SRD1, involved in the processing of pre-rRNA to mature rRNA. The SRD1 gene was identified by selecting for second-site suppressors of the previously described rrp1-1 mutation. The rrp1-1 mutation causes temperature-sensitive growth, a conditional defect in processing of 27S pre-rRNA to mature 25S rRNA, and a nonconditional increase in sensitivity to several aminoglycoside antibiotics. All srd1 alleles identified are recessive and apparently specific to the rrp1-1 mutation. Although a mutation of SRD1 suppresses the pre-rRNA processing defect, drug sensitivity and thermolethality of a point mutation of RRP1, it is unable to suppress a rrp1-disruption allele. We suggest that the SRD1 gene product either interacts with or regulates the RRP1 product.

Tumor uptake and elimination of 2',2'-difluoro-2'-deoxycytidine (gemcitabine) after deoxycytidine kinase gene transfer: correlation with in vivo tumor response.

PURPOSE: We hypothesized that tumor uptake and elimination of 2',2'-difluoro-2'-deoxycytidine/2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (dFdCyd/dFdCTP) would be altered after dCK gene transfer and that this change would result in an enhanced cytotoxic effect. To test this hypothesis, we examined dFdCyd/dFdCTP uptake and clearance in HT-29 human colon carcinoma xenografts in nude mice by high-performance liquid chromatography (HPLC) and fluorine-19 magnetic resonance spectroscopy (F-19 MRS). EXPERIMENTAL DESIGN: HT-29 tumors were grown from cells infected with either the retroviral vector alone (LNPO-LacZ) or vector containing the dCK gene (LNPO-dCK). HPLC and F-19 MRS analyses were performed after a single 160 mg/kg i.p. injection of dFdCyd. Tumor response was determined in animals receiving a similar dosing schedule of dFdCyd. RESULTS: HPLC experiments revealed an increased tumor accumulation of dFdCTP in xenografts overexpressing dCK compared with wild-type controls (P < or = 0.05). dFdCTP in the dCK-infected tumors was easily identified at 24 h postinjection. Conversely, no dFdCTP could be detected in the control xenografts 14 h postinjection. Subsequent F-19 MRS experiments confirmed an altered uptake, revealing a 2.5-fold greater accumulation of dFdCyd/dFdCTP in the dCK xenografts. Whereas a modest tumor growth delay was observed in the wild-type tumors receiving dFdCyd, dCK xenografts demonstrated a marked tumor growth delay following treatment (P < or = 0.05). CONCLUSIONS: These data support the hypothesis that increased expression of dCK cDNA in HT-29 xenografts results in an enhanced dFdCTP accumulation and prolonged elimination kinetics, and ultimately a potentiated in vivo tumor response to dFdCyd. Related to these effects, changes in the overall tumor metabolism of dFdCyd/dFdCTP was detectable by noninvasive F-19 MRS. These data are relevant to future preclinical and clinical studies evaluating dCK gene transfer and dFdCyd therapy.

SRD1, a S. cerevisiae gene affecting pre-rRNA processing contains a C2/C2 zinc finger motif.

The Saccharomyces cerevisiae genes, RRP1 and SRD1, are involved in processing rRNA precursor species to mature rRNAs. We reported previously that the rrp1-1 mutation caused temperature-sensitive lethality, hypersensitivity to aminoglycoside antibiotics, and defective processing of 27S pre-rRNA to 25S and 5.8S mature rRNAs. A second-site suppressor of the rrp1-1 mutation, srd1, corrects all three rrp1 mutant phenotypes. In order to learn more about the roles of the SRD1 and RRP1 genes in rRNA processing, we cloned and characterized the SRD1 gene. We identified an ORF, YCR18C, that complements srd1-2 suppression of rrp1-1. The DNA is physically located at the region of chromosome III where SRD1 has been genetically mapped. SRD1 encodes a putative 225 amino acid, 26 kDa protein containing a C2/C2 zinc finger motif that is also found in some transcription regulators and the eIF-2 beta translation initiating factors. The similarity of SRD1 to transcription regulators led us to test the model that srd1 mutations suppress rrp1 defects by altering the level of the RRP1 transcript. However, we found that SRD1 has no detectable effect on the steady state levels of RRP1 mRNA. We describe alternative models to explain the role of Srd1p in pre-rRNA processing.

Modulators of cyclic AMP systems.

On the basis of the data reported here, one may conclude that although many agents that act in the central nervous system are modulators of the action of cyclic AMP, it is difficult to establish a direct connection between the pharmacologic activity and the levels of cyclic AMP in the brain. This lack of interrelation applies to the benzodiazepines as well as to the pyrazolopyridines. The data for members of the latter group are somewhat frustrating in this regard, since an excellent correlation has been shown to exist between the potency of inhibition of PDE and activity in the antianxiety test. In measurements of steroidogenesis in the isolated adrenal cell, the correlation between activity in vito and the conflict assay is even better. The data presented here and reported elsewhere (Shimizu et al., 1974; Kelly et al., 1974; Mayer and King, 1974; King and Mayer, 1974) provide evidence that agents that act as inhibitors of PDE in cell-free systems exert their influence on cyclic AMP in tissue slices of the brain of guinea pigs by mechanisms that seem not to be related to an effect on PDE. Papaverine, and possibly chlordiazepoxide, may act by releasing agonists that, in turn, stimulate the accumulation of cyclic AMP. This activity is blocked bo other inhibitors of PDE, such as theophyline. Results obtained by the use of platelets are refreshingly clear. Inhibition of aggregation has been shown to occur when the level of cyclic AMP is raised, and a suggestive exists that the most potent inhibitors of platelet PDE are the best potentiators of the action of PGE1 in blocking aggregation. The study utilizing drugs collected from a large number of therapeutic classes makes clear that it is difficult to attribute the mechanism of action for any of the classes studied to modulation of cyclic AMP. An unexpected finding of this study, however, was the fact that pharmacologic agents include an unusually large number of inhibitors of PDE as compared with agents chosen at random. This finding provides a powerful tool for the biochemical pharmacologist who is examining large numbers of compounds in the search for potential drugs.