Overexpression of a Heterologous Thymidine Kinase Delays Apoptosis Induced by Factor Deprivation and Inhibitors of Deoxynucleotide Metabolism

Oliver, F. Javier; Collins, Mary; López‐Rivas, Abelardo

doi:10.1074/jbc.272.16.10624

Cited by 28 publications

(20 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This imbalance is accompanied by reduced activity of thymidine kinase, an enzyme controlling dNTP synthesis, and followed by apoptosis (Oliver et al, 1996). Strikingly, overexpression of a heterologous thymidine kinase protects cells from apoptosis through IL-3 removal (Oliver et al, 1997). Our analyses showed that thymidine kinase is repressed in either of the apoptotic pathways, consistent with a more general anti-apoptotic role.…”

Section: Genes Regulated In Both Apoptosis-induction Pathwayssupporting

confidence: 65%

Comparative microarray analysis of gene expression during apoptosis-induction by growth factor deprivation or protein kinase C inhibition

et al. 2000

View full text Add to dashboard Cite

Section: Genes Regulated In Both Apoptosis-induction Pathwayssupporting

confidence: 65%

Comparative microarray analysis of gene expression during apoptosis-induction by growth factor deprivation or protein kinase C inhibition

et al. 2000

View full text Add to dashboard Cite

“…This is in agreement with Chung et al (37), who reported increased expression of TK in 5-FU-resistant gastric cancer cells. Furthermore, Oliver et al (40) showed that overexpression of a heterologous TK gene protected murine BAF3 cells from apoptosis induced by inhibitors of nucleotide synthesis, such as methotrexate or fluorodeoxyuridine. The authors suggest that salvaging of thymidine by TK may compensate for inhibition of de novo thymidylate synthesis and thereby abrogate thymineless death.…”

Section: Discussionmentioning

confidence: 99%

Characterization of p53 Wild-Type and Null Isogenic Colorectal Cancer Cell Lines Resistant to 5-Fluorouracil, Oxaliplatin, and Irinotecan

Boyer¹,

McLean²,

Aroori³

et al. 2004

Clinical Cancer Research

195

144

View full text Add to dashboard Cite

To elucidate mechanisms of resistance to chemotherapies currently used in the first-line treatment of advanced colorectal cancer, we have developed a panel of HCT116 p53 wild-type (p53 ؉/؉ ) and null (p53 ؊/؊ ) isogenic colorectal cancer cell lines resistant to the antimetabolite 5-fluorouracil (5-FU), topoisomerase I inhibitor irinotecan (CPT-11), and DNA-damaging agent oxaliplatin. These cell lines were generated by repeated exposure to stepwise increasing concentrations of each drug over a period of several months. We have demonstrated a significant decrease in sensitivity to 5-FU, CPT-11, and oxaliplatin in each respective resistant cell line relative to the parental line as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, with increases in IC 50 (72 h) concentrations ranging from 3-to 65-fold. Using flow cytometry, we have also demonstrated compromised apoptosis and cell cycle arrest in 5-FU-, oxaliplatin-, and CPT-11-resistant cell lines compared with the parental lines after exposure to each drug. In addition, we found that resistance to 5-FU and oxaliplatin was higher in parental p53 ؊/؊ cells compared with parental p53 ؉/؉ cells, with an ϳ5-fold increase in IC 50 (72 h) for each drug. In contrast, the IC 50 (72 h) doses for CPT-11 were identical in the p53 wild-type and null cell lines. Furthermore, apoptosis after treatment with 5-FU and oxaliplatin, but not CPT-11, was significantly reduced in parental p53 ؊/؊ cells compared with parental p53 ؉/؉ cells. These data suggest that p53 may be an important determinant of sensitivity to 5-FU and oxaliplatin but not CPT-11. Using semiquantitative reverse transcription-PCR, we have demonstrated down-regulation of thymidine phosphorylase mRNA in both p53 ؉/؉ and p53 ؊/؊ 5-FU-resistant cells, suggesting that decreased production of 5-FU active metabolites may be an important resistance mechanism in these lines. In oxaliplatin-resistant cells, we noted increased mRNA levels of the nucleotide excision repair gene ERCC1 and ATP-binding cassette transporter breast cancer resistance protein. In CPT-11-resistant cells, we found reduced mRNA levels of carboxylesterase, the enzyme responsible for converting CPT-11 to its active metabolite SN-38, and topoisomerase I, the SN-38 target enzyme. In addition, we noted overexpression of breast cancer resistance protein in the CPT-11-resistant lines. These cell lines are ideal tools with which to identify novel determinants of drug resistance in both the presence and absence of wild-type p53.

show abstract

“…Because TTP is an allosteric effector of ribonucleotide reductase, imbalances in the TTP pool disturb the supply of both purines and pyrimidines for DNA synthesis and repair. In turn, imbalanced deoxynucleoside triphosphate (dNTP) pools increase the mutation rate and probability of carcinogenesis (1)(2)(3).…”

mentioning

confidence: 99%

Valine, Not Methionine, Is Amino Acid 106 in Human Cytosolic Thymidine Kinase (TK1)

Berenstein

Christensen

Kristensen

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

(t1 ⁄2 at 15°C is 41 and 392 min, respectively). Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.The human cytosolic thymidine kinase, TK1 (EC 2.7.1.21), is a key enzyme in the salvage synthesis of TMP from thymidine. Intracellular TMP is quickly phosphorylated to TDP and TTP. Because TTP is an allosteric effector of ribonucleotide reductase, imbalances in the TTP pool disturb the supply of both purines and pyrimidines for DNA synthesis and repair. In turn, imbalanced deoxynucleoside triphosphate (dNTP) pools increase the mutation rate and probability of carcinogenesis (1-3).TK1 is a cell cycle-regulated enzyme. Its activity fluctuates with DNA synthesis, being high in dividing and malignant cells and low in quiescent cells (4, 5). The expression of TK1 is meticulously controlled on the transcriptional and post-transcriptional level (6, 7). At the enzymatic level, ATP, besides being a cosubstrate, has been shown to be a regulator of the catalytic activity of TK1 (8). Thus, exposure to ATP induces a reversible enzyme concentration-dependent transition from a low thymidine affinity dimer of about 50 kDa (K m ϭ 15 M) to a high affinity tetramer (K m ϭ 0.7 M). To further investigate the effect of ATP, we constructed a pET3a-TK1 plasmid (9) containing the amino acid coding region of TK1 cDNA from the pTK11 plasmid of Bradshaw and Deininger (10), who had used SV40 transformed human fibroblasts as the mRNA source. We expressed the resulting recombinant TK1 (rTFi-TK1) in Escherichia coli, and purified and characterized the enzyme. To our surprise we found that the enzymatic properties of rTFi-TK1 differed markedly from those of the endogenous Ly-TK1 with respect to the regulatory effect of ATP (9). Irrespective of preexposure to ATP, the recombinant rTFi-TK1 was a permanent tetramer of about 100 kDa with high affinity to thymidine with a K m value about 0.4 M (9). At that time, we assumed that the amino acid sequences of rTFi-TK1 and Ly-TK1 were identical and explained the divergent properties of rTFi-TK1 by the absence of post-translational modification of TK1 when expressed in E. coli (9). Because the pET3a-TK1 expression system was not satisfactory in terms of amount of TK1 protein produced, we constructed another expression plasmid, pGEX-2T-LyTK1. Here, the amino acid coding region of TK1 from normal human lymphocytes was cloned into the pGEX-2T vector, and the recombinant TK1 was expressed as an isopropyl-1-thio-␤-D-galactopyranoside-inducible glutathione S-transferase fusion protein. In contrast to the findings with rTFi-TK1, our preliminary kinetic experiments showed that the recombinant lymphocyte TK1 (rLy-TK1) 1 behaved essentially as the endogenous lymphocyte enzyme, Ly-TK1, regarding kinetic and oligomerization properties. Therefore, absence of posttranslational modification of rTFi-TK1 in E. coli cannot ex...

show abstract

Overexpression of a Heterologous Thymidine Kinase Delays Apoptosis Induced by Factor Deprivation and Inhibitors of Deoxynucleotide Metabolism

Cited by 28 publications

References 43 publications

Comparative microarray analysis of gene expression during apoptosis-induction by growth factor deprivation or protein kinase C inhibition

Comparative microarray analysis of gene expression during apoptosis-induction by growth factor deprivation or protein kinase C inhibition

Characterization of p53 Wild-Type and Null Isogenic Colorectal Cancer Cell Lines Resistant to 5-Fluorouracil, Oxaliplatin, and Irinotecan

Valine, Not Methionine, Is Amino Acid 106 in Human Cytosolic Thymidine Kinase (TK1)

Contact Info

Product

Resources

About