Escherichia coli thymidylate synthase expression protects human cells from the cytotoxic effects of 5-fluorodeoxyuridine more effectively than human thymidylate synthase overexpression.

In this study, we compared the relative abilities of human thymidylate synthase (hTS) and Escherichia coli thymidylate synthase (eTS) expression to confer resistance to the cytotoxic effects of treatment with the TS inhibitor 5-fluorodeoxyuridine (FdUrd). G418-selected clones expressing either form of the protein were significantly more resistant than the lacZ-expressing clone, VALZ2, to FdUrd-induced cytotoxicity. Although eTS-expressing clones expressed 2- to 3-fold more TS protein than hTS-overexpressing clones, the representative eTS-expressing clone, VAEG8, and hTS-overexpressing clone, VAHGC, were equally sensitive to an FdUrd-induced loss of clonogenicity; in addition, a large fraction of either form of exogenously expressed TS appeared to be inactive in the intact cell. The clones differed, however, in their responses to leucovorin (LV). Although LV significantly enhanced FdUrd-induced TS inhibition, growth inhibition, and cytotoxicity in VAHGC cells, it had no effect on these parameters in VAEG8 cells. These results suggest that eTS may more efficiently confer resistance to FdUrd plus LV when expressed for the purposes of a "host protection" strategy in vivo.

Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA.

Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5'-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434-634. In this paper, a 70 nt RNA sequence, corresponding to nt 480-550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434-634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5'-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434-634 or p644/TS480-550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT-PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480-550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434-634 and p644/TS480-550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.

N-Nitrosodimethylamine-mediated cytotoxicity in a cell line expressing P450 2E1: evidence for apoptotic cell death.

N-Nitrosodimethylamine (NDMA) is an acute hepatotoxin and potent carcinogen. The metabolic activation of NDMA to reactive metabolites is a critical step for the expression of its toxic and carcinogenic potential. We have previously demonstrated a strong correlation between methylation of cellular macromolecules and NDMA-mediated cytotoxicity, and we have demonstrated that reactive oxygen species may partially contribute to the toxic effects in P450 2E1-expressing cells. The mode of cell death in NDMA-treated monolayer cultures exhibited the following characteristics: (i) condensation of nuclear chromatin as demonstrated by using Hoechst 33258 staining, (ii) DNA fragmentation as detected by combining pulsed field and conventional agarose gel electrophoresis, and (iii) DNA double strand breaks determined by using the in situ terminal deoxynucleotidyl transferase assay and flow cytometric analysis. These results indicate that reactive metabolites of NDMA trigger activation of the signal pathway for apoptotic cell death in these P450-expressing cells. The NDMA-mediated cell death was partially prevented by the endonuclease inhibitor, aurintricarboxylic acid, as well as the caspase inhibitors, acetyl-Asp-Glu-Val-Asp-CHO and acetyl-Tyr-Val-Ala-Asp-CHO. The cell cycle distribution was altered in NDMA-treated cells resulting in an increase in the G2/M phase and a decrease in the G1 phase. Our results suggest that DNA degradation, the inability to complete DNA repair, the biochemical events associated with G2/M arrest, and the process of apoptotic death all result from P450 2E1-catalyzed metabolism of NDMA.

Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells.

PURPOSE: We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity. In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors. In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity. METHODS: Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones. HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity. RESULTS: Following a 24-h treatment with 100 microM CB3717 or 500 nM methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells. DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h. However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line. In addition, expression of dutE in two HuTu80 clones, 80 C and 80 K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity. CONCLUSIONS: We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line. The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines.

Prevention of fluorodeoxyuridine-induced cytotoxicity and DNA damage in HT29 colon carcinoma cells by conditional expression of wild-type p53 phenotype.

We have examined the effects of conditionally expressing wild-type p53 activity in HT29 cells on DNA damage and cytotoxicity caused by exposure to fluorodeoxyuridine (FdUrd). Expression of wild-type p53 phenotype for 24 hr before FdUrd treatment provided HT29 cells with virtually complete protection from cytotoxicity caused by this drug. In addition, wild-type p53 expression also prevented FdUrd-induced DNA double-strand breaks and, unexpectedly, single-strand breaks in parental (mature) DNA. Temporary expression of wild-type p53 activity in the absence of drug treatment caused some loss of clonogenicity, although the magnitude of this cytotoxic effect was small compared with the level of cell kill obtained by treatment with cytotoxic drugs for similar periods of time, indicating that HT29 cells are not highly sensitive to induction of programmed cell death by wild-type p53. Because these observations conflict with previously suggested models for FdUrd-induced damage to parental DNA, we propose an alternative model to explain how incorporation of uracil into nascent DNA might result in single-strand breaks in the opposite (parental) strand and how these breaks might be converted to the double-strand breaks that produce cell death.

Induction of resistance to fluorodeoxyuridine cytotoxicity and DNA damage in human tumor cells by expression of Escherichia coli deoxyuridinetriphosphatase.

Recent studies from our laboratory suggested that, in some human colorectal tumor cell lines, sensitivity to fluorodeoxyuridine may depend upon the extent of dUTP accumulation that occurs following drug treatment and that elevation of dUTPase activity might be the basis for some instances of resistance to fluoropyrimidines. To test this model, we expressed Escherichia coli dUTPase in an established human tumor cell line (HT29) and measured the effect of this manipulation on response to fluorodeoxyuridine. As predicted, HT29 derivatives containing dUTPase activity 4-5-fold higher than controls were protected from fluorodeoxyuridine-induced loss of clonogenicity and from formation of DNA double strand breaks. These data provide the first direct evidence that alteration in a component of the uracil misincorporation/misrepair pathway can confer resistance to fluoropyrimidines in human tumor cells.

Transient transfection studies of secretion in bovine chromaffin cells and PC12 cells. Generation of kainate-sensitive chromaffin cells.

We have developed a transient transfection method to measure protein secretion from non-dividing, primary bovine chromaffin cells and from the continuous cell line, PC12. A plasmid coding human growth hormone (GH) was expressed in sufficient amounts in bovine chromaffin and PC12 cells to allow precise measurements of secretion from the small fraction (less than 1%) of transfected cells in a dish. GH was secreted in a similar proportion to endogenous catecholamine upon nicotinic stimulation, depolarization with elevated K+, and upon permeabilization with digitonin and subsequent stimulation with micromolar Ca2+. GH in homogenates from GH-transfected chromaffin cells cosedimented with catecholamine on discontinuous sucrose gradients. The data indicate that transiently expressed human GH in chromaffin and PC12 cells is localized predominantly in secretory vesicles in the regulated secretory pathway. With transient transfection there is a high probability of coexpression in the same cell of two plasmids which are cotransfected. Coexpression of a plasmid for GH and a plasmid for the non-N-methyl-D-aspartate glutamate receptor, GluR1, created chromaffin cells in which Ca(2+)-dependent GH secretion could be stimulated by the glutamatergic agonist kainate. The ability to coexpress a plasmid of interest with a plasmid for GH will allow the investigation of the role of other cloned proteins in the regulated secretory pathway in differentiated, non-dividing cells.