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K D Danenberg

Publications and source records attributed to K D Danenberg.

At least 37 records · Page 2Linked to original sources
PubMed1999

CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression.

The molecular basis of aberrant hypermethylation of CpG islands observed in a subset of human colorectal tumors is unknown. One potential mechanism is the up-regulation of DNA (cytosine-5)-methyltransferases. Recently, two new mammalian DNA methyltransferase genes have been identified, which are referred to as DNMT3A and DNMT3B. The encoded proteins differ from the predominant mammalian DNA methyltransferase DNMT1 in that they have a substantially higher ratio of de novo to maintenance methyltransferase activity. We have used a highly quantitative 5' nuclease fluorogenic reverse transcription-PCR method (TaqMan) to analyze the expression of all three DNA methyltransferase genes in 25 individual colorectal adenocarcinoma specimens and matched normal mucosa samples. In addition, we examined the methylation patterns of four CpG islands [APC, ESR1 (estrogen receptor), CDKN2A (p16), and MLH1] to determine whether individual tumors show a positive correlation between the level of DNA methyltransferase expression and the frequency of CpG island hypermethylation. All three methyltransferases appear to be up-regulated in tumors when RNA levels are normalized using either ACTB (beta-actin) or POLR2A (RNA pol II large subunit), but not when RNA levels are normalized with proliferation-associated genes, such as H4F2 (histone H4) or PCNA. The frequency or extent of CpG island hypermethylation in individual tumors did not correlate with the expression of any of the three DNA methyltransferases. Our results suggest that deregulation of DNA methyltransferase gene expression does not play a role in establishing tumor-specific abnormal DNA methylation patterns in human colorectal cancer.

Adenocarcinoma↗
PubMed1998

Intercellular communication mediates the bystander effect during herpes simplex thymidine kinase/ganciclovir-based gene therapy of human gastrointestinal tumor cells.

A bystander effect is described when nontransduced or genetically unmodified cells are killed during death of genetically modified tumor cells transduced with a suicide gene. The "bystander effect" greatly enhances the efficacy of the herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy approach for cancer. The mechanism of the bystander effect is controversial. In this study, we examined the role of intercellular gap junction communication (GJIC) for the bystander effect in human gastrointestinal tumor cells. Our results show that the extent of the bystander effect varied amongst the tumor cell lines; pancreatic cancer cells BXPC-3 exhibited excellent bystander effects in vitro and in vivo studies whereas other gastrointestinal tumor cell lines such as pancreatic cancer cells MIAPACA-2, and colon cancer cells HT-29 showed poor bystander effects. Bystander effects were only found in the presence of cell-to-cell contact. The extent of the bystander effect was independent of the level of HSV-TK activity in the transduced tumor cells and was correlated with GJIC as demonstrated by an in vitro dye-transfer assay. Expression of the mRNA levels of gap junction protein connexin 43 was 8- to 26-fold or greater and connexin 26 gene expression was 2- to 229-fold greater in BXPC-3 cells compared to HT-29, MIAPACA-2, and PANC3 cells. Our results suggest that intercellular communication is essential for the bystander effect. The correlation between GJIC and the extent of the bystander effect suggest a role for GJIC in mediating the bystander effect. Analysis of tumors for GJIC or expression of gap junction proteins may identify the subset of patients suitable for gene therapy with the HSV-TK/GCV approach.

Animals↗
PubMed1998

ERCC1 mRNA levels complement thymidylate synthase mRNA levels in predicting response and survival for gastric cancer patients receiving combination cisplatin and fluorouracil chemotherapy.

PURPOSE: We have previously shown that relative thymidylate synthase (TS) mRNA levels in primary gastric adenocarcinomas treated with fluorouracil (5-FU) and cisplatin are inversely associated with response and survival. This is a presumed function of TS as a target for 5-FU activity. We now test the hypotheses that the relative mRNA level of the excision repair cross-complementing (ERCC1) gene is inversely associated with response and survival as an independent function of cisplatin efficacy. PATIENTS AND METHODS: Patients had intact, untreated, primary gastric adenocarcinoma cancer and were evaluated for eligibility on a preoperative cisplatin infusion-5-FU protocol. cDNA, derived from primary gastric tumors before chemotherapy, was used to determine ERCC1 mRNA levels, expressed as the ratio of polymerase chain reaction (PCR) product of the ERCC1 gene and the beta-actin gene. RESULTS: The median ERCC1 mRNA level from 38 primary gastric cancers (33 assessable for response) was 5.8 x 10(-3) (range, 1.8 x 10(-3) to 19.5 x 10(-3)). Of 17 responding patients, 13 (76%) were less than or equal to 5.8 x 10(-3) and four were greater than 5.8 x 10(-3) (P = .003). The median survival for patients with ERCC1 mRNA levels less than or equal to 5.8 x 10(-3) has not been reached, whereas for those greater than 5.8 x 10(-3) it was 5.4 months (P = .034). The median TS mRNA level, 3.7 x 10(-3) (range, 0.9 to 18.9) also segregated responsive versus resistant tumors (P = .024). With both ERCC1 and TS mRNA levels below their medians, 11 of 13 patients (85%) responded; with both ERCC1 and TS mRNA levels above their medians, two of 10 patients (20%) responded (P = .003). CONCLUSION: Considered separately, either ERCC1 or TS mRNA levels in a primary gastric adenocarcinoma has a statistically significant relationship to response. ERCC1 mRNA levels have a statistically significant association with survival; in this cohort TS mRNA levels did not reach statistically significant association with survival as in our previous publication. Whether these molecular parameters are independent of each other as predictors of outcome remains to be determined.

Adenocarcinoma↗
PubMed1998

Higher levels of thymidylate synthase gene expression are observed in pulmonary as compared with hepatic metastases of colorectal adenocarcinoma.

PURPOSE: It has been observed previously that the pulmonary metastases of colorectal adenocarcinoma are less responsive to therapy with fluorouracil (FUra) as compared with other sites of metastasis (liver, local). To investigate the basis of this chemoresistance, the levels of thymidylate synthase (TS) mRNA and protein were measured, as TS expression has been shown to be predictive of response to therapy in colorectal cancer. MATERIALS AND METHODS: Tumors were obtained from 19 patients with metastatic colorectal cancer (12 hepatic and seven pulmonary). TS expression was measured by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and TS protein levels were measured by Western blotting. The presence of TS amplification was assessed by Southern blotting. Levels of p53 protein were determined using immunohistochemistry. RESULTS: TS mRNA expression was shown to be significantly higher in the pulmonary metastases (mean TS/beta-actin ratio, 19.7; n = 7) as compared with the hepatic metastases (mean TS/beta-actin ratio, 4.7; n = 11) of colorectal cancer. Lower TS expression was observed in patients with hepatic metastases who had received prior FUra versus patients who had not been treated. High levels of TS expression in some samples was associated with low-level (two to three gene copies) increases in TS gene copy numbers and this was observed more frequently in the pulmonary metastatic samples. The increased gene copy numbers occurred both in samples with wild-type p53 and those with mutant p53 tumor-suppressor gene as determined by immunohistochemistry. CONCLUSION: High levels of TS enzyme may be the basis of the lack of response of pulmonary metastases to FUra treatment.

Adenocarcinoma↗
PubMed1998

p53 and thymidylate synthase expression in untreated stage II colon cancer: associations with recurrence, survival, and site.

We initiated a retrospective study to determine whether p53 status and thymidylate synthase (TS) protein expression in primary colon tumors influence recurrence and survival for patients with stage II colon cancer. Tumor specimens from 45 consecutive untreated patients with stage II colon cancer were examined for p53 and TS protein expression using immunohistochemistry. The median follow-up was 5.1 years. Eighteen patients had left-sided tumors, and 27 had right-sided tumors. Fourteen of 45 patients (31%) developed recurrence. p53 overexpression was detected in the tumors of 18 patients (40%); 10 patients (55%) with p53 overexpression recurred; and 4 of 27 (15%) without evidence of p53 overexpression recurred (P = 0.002). High TS expression was detected in the tumors of 16 patients (36%): 8 patients (50%) with high TS expression recurred, and 6 patients (21%) with low TS expression recurred (P = 0.027). Patients with p53 overexpression had a significantly poorer survival than did those patients without p53 overexpression (P < 0.001). High TS expression was associated with poor survival (P = 0.004). p53 overexpression and high TS expression were significantly associated with left-sided tumors (P = 0.003 and P = 0.022). Thirteen of 16 patients (81%) with high TS expression also overexpressed p53, and 24 of 29 patients (81%) with low TS expression did not manifest p53 overexpression (P < 0.001). p53 and TS expression in primary stage II colon cancer are associated and appear to influence recurrence and survival. In this pilot study, left-sided tumors demonstrate significantly more p53 overexpression and significantly higher TS expression than do right-sided tumors, which may explain the significantly poorer survival for patients with left-sided tumors.

Adult↗
PubMed1998

p53 point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: an analysis of response and survival.

Recent studies suggest that there may be a strong correlation between the p53 status of a tumor and a patient's response to chemotherapy. Therefore, we determined p53 status in 36 patients with disseminated colorectal cancer by cDNA sequencing and immunohistochemical staining, as well as by the gene expression level of thymidylate synthase (TS), the target enzyme of 5-fluorouracil (5-FU), by reverse transcription-PCR. Ten patients (28%) experienced a clinical response to 5-FU chemotherapy. Overall, TS expression and response to chemotherapy were associated: 9 of 18 (50%) patients with TS < or = 3.0 x 10(-3) responded, compared to 1 of 18 (6%) patients with TS > 3.0 x 10(-3) (P = 0.003). p53 mutations were found in 21 of 36 patients (58%) using cDNA cycle sequencing, and p53 protein overexpression was found in 20 of 32 patients (62%) using immunohistochemistry staining. Overall p53 status and response to chemotherapy were associated: 5 of 10 (50%) patients with wild-type p53 or negative p53 staining experienced a response, but only 5 of 26 (19%) patients with mutant p53 or p53 overexpression responded. TS expression, but not expression of p53, was significantly associated with overall survival (P = 0.002). Patients with wild-type p53 had significantly lower TS levels compared to patients with mutated p53 (P = 0.044). In this study, we also present data linking specific p53 point mutations to TS expression levels and resistance to 5-FU. Although the number of patients is relatively small, these results identify p53 status and TS gene expression as associated with response in disseminated colorectal cancer; independent studies are needed to confirm these findings and to provide information leading to a better understanding of the role of 5-FU-based chemotherapy in the treatment of colorectal cancer.

Adult↗
PubMed1998

Restoration of wild-type p53 activity in p53-null HL-60 cells confers multidrug sensitivity.

HL-60 cells that stably express transfected wild-type (wt) p53 were used to determine whether restoration of wt p53 increased the chemosensitivity of cells that normally lack p53 activity. The wt p53 HL-60 transfectants (SN3 cells) were more sensitive than the parental (S) cells to a number of common anticancer drugs representing various mechanisms of action, whereas HL-60 cells transfected with p53 genes mutated at codons 248 and 143 were not sensitized. The sensitization ratio due to the transfected wt p53 varied from about 2-fold for cisplatin to over 50-fold for thymidine. Cells treated with the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FdUrd) were used to study changes in various p53-associated gene expressions. A higher percentage of apoptotic cells among the SN3 cells was observed than among the S cells at each concentration of FdUrd. The S cells had undetectable levels of bax and high levels of bcl-2, whereas the SN3 cells had undetectable levels of bcl-2 levels and appreciable basal levels of bax. After FdUrd treatment of SN3 cells, both p53 and bax levels increased, but the induction of bax was faster than that of p53 and paralleled the appearance of apoptotic DNA laddering. FdUrd treatment induced p21 expression and increased the G1 fraction of the SN3 cells but did not induce p21 or change the phase distribution in the S cells. FdUrd treatment also induced the expression and phosphorylation of cyclin D1 in the SN3 cells but not in the S cells. These results show that transfected wt p53 confers multidrug sensitivity to HL-60 cells by re-adjustment of the expressions of apoptosis genes and displays other properties characteristic of endogenously originated wt p53.

Antineoplastic Agents↗
PubMed1998

Desensitization and sensitization of cells to fluoropyrimidines with different antisenses directed against thymidylate synthase messenger RNA.

Previous studies have shown that the cytotoxicity of fluoropyrimidines is mediated, in large part, by inhibition of the enzyme thymidylate synthase (TS). The aim of this study was to determine whether the chemosensitivity of human cancer cells to fluoropyrimidines could be increased by decreasing TS expression with antisense oligodeoxyribonucleotides (ODNs). ODNs (18-mers) targeted at the AUG translational initiation site of TS mRNA inhibited translation in a sequence- and dose-dependent manner in a rabbit reticulocyte lysate in vitro translation system. Treatment of human colon cancer HT-29 cells with antisense ODNs decreased TS catalytic activity in the cells in a dose-dependent manner over a short period, but the longer-term effect of the TS antisense ODN treatment was actually to increase the amount of TS in the cells and to decrease their sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd). However, when human nasopharyngeal cancer KB31 cells were transfected with a plasmid (pHaMAGRP) construct containing the TS antisense fragment (+ 1 to + 422) under the control of a glucose-regulated promoter, the expression of both TS protein and TS catalytic activity was decreased by nearly 30% (P = 0.014), and sensitivity of these cells to FdUrd was enhanced by approximately 8-fold (P = 0.021). No changes in the levels of expression of TS protein or FdUrd-associated cytotoxicity were observed in control, vector-transfected cells. No change was observed in the sensitivity of transfected cells toward either cisplatin or Adriamycin. These results show that the level of expression of TS in human malignant cells can be down-regulated with antisense TS RNA, and their sensitivity to fluoropyrimidines can, thereby, be increased.

Animals↗
PubMed1997

Thymidylate synthase is a predictor for response and resistance in hepatic artery infusion chemotherapy.

The value of intratumoral thymidylate synthase (TS) quantitation as a predictive parameter for hepatic artery infusion (HAI) chemotherapy in patients with colorectal liver metastases was investigated. Relative TS mRNA levels were determined in 29 tumor samples using a quantitative RT-PCR amplification method. The median level of expression was 3.0 x 10(-3) (no units) and varied considerably among the tumors over a range of 135-fold. Patients with low TS levels were 4.1-fold more likely to respond (P < 0.03) compared to patients with high TS levels. Our results indicate that TS quantitation is a valuable predictive marker for tumor response to HAI therapy.

Adult↗
PubMed1996

Thymidylate synthase mRNA level in adenocarcinoma of the stomach: a predictor for primary tumor response and overall survival.

PURPOSE: We tested the hypothesis that polymerase chain reaction (PCR) quantitation of the enzyme thymidylate synthase (TS) within a primary adenocarcinoma of the stomach, has an inverse relationship to response and survival for patients who receive fluorouracil (5FU)-based chemotherapy. PATIENTS AND METHODS: Before systemic chemotherapy, the genetic expression of TS (TSmRNA level) was determined using a PCR method. Gene expression was calculated by determining the ratio between the amount of radiolabeled PCR product with the linear amplification range of the TS gene and the beta-actin gene. Chemotherapy consisted of two cycles of protracted infusion (PI) 5FU 200 mg/m2/d administered for 3 weeks with leucovorin 20 mg/m2/w. Cisplatin 100 mg/m2 was administered on day 1. RESULTS: Sixty-five patients with primary gastric cancer had a median TS mRNA level of 4.6 x 10(-3) (range, 0.9 to 20.1 x 10(-3)). Thirty-five percent of patients had measurable responses in their primary tumors. The mean gastric cancer TSmRNA level in responding and resistant patients is statistically significant (P < .001). The median survival time was 43+ months for treated patients with TSmRNA levels less than the median and 6 months for those with TS m-RNA levels greater than the median (P = .003). CONCLUSION: The genetic expression of TS (TSmRNA level) influences response to 5FU-based chemotherapy and survival for a cohort of patients with primary gastric cancer. Confirmation of these data could lead to therapeutic decisions based on specific molecular properties within a tumor.

Adenocarcinoma↗
PubMed1995

Identification of mutations by RNA conformational polymorphism "bar code" analysis.

DNA single-strand conformational polymorphism (SSCP) analysis is widely used for detection of point mutations in clinical specimens. Performing SSCP analysis with cRNA instead of DNA has been shown to improve mutation detection frequency. RNA can exist in numerous meta-stable conformations, which appear as patterns of bands on nondenaturing electrophoresis gels. Single base mutations can cause not only mobility shifts of major bands, but also loss of some conformations and appearance of new conformations. Unique RNA SSCP patterns associated with specific base sequences in many cases allow visual identification of point mutations. However, in some cases, the RNA SSCP pattern of a single base change in a sequence is not sufficiently different for a positive identification of the mutation. Improvement in the detection capability of RNA SSCP was obtained by adding 3'-deoxynucleotides to the transcription reaction. The presence of chain-terminating nucleotides in the transcription reaction formed numerous new RNA fragments, thereby generating complex band patterns ("bar codes") unique to each RNA sequence. This method was applied to analyzing p53 mutations in patients with colon cancer.

Base Sequence↗
PubMed1995

Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors.

Thymidylate synthase (TS) is the target enzyme for 5-fluorouracil (5-FU). We have correlated TS protein and gene expression with the response in patients with colorectal (n = 9) and gastric cancer (n = 12) treated with infusional 5-FU plus leucovorin (LV) or infusional 5-FU/LV and cisplatin, respectively. TS protein expression was analyzed by Western blot using TS106 monoclonal antibody and densitometry scanning. TS gene expression was measured by PCR analysis using beta-actin as an internal standard and expressed as a TS:beta-actin mRNA ratio. A close linear relationship was noted between TS protein expression and TS gene expression (r2 = 0.60) for the 21 tumor samples analyzed. TS immunohistochemical staining on 15 of the 21 samples revealed that the TS staining intensity correlated closely with TS protein and mRNA expression. In two biopsy samples, TS protein levels and TS gene expression did not correlate; however, one of these exhibited a focal TS staining pattern. Both the TS protein level and TS gene expression were significantly associated with response to 5-FU-based therapy. Patients with responsive disease had a mean TS protein level of 0.17 +/- 0.03 arbitrary units (range, 0.05 to 0.38), whereas in patients whose tumors did not respond, the mean TS protein level was significantly higher 0.60 +/- 0.09 (range, 0.06 to 1.01; P < 0.01). A similar pattern was noted with TS gene expression. In patients with responsive disease, the mean TS:beta-actin gene ratio was 1.36 +/- 0.3 (range, 0.5-3.3 x 10(-3). In contrast, biopsies from patients with unresponsive disease had a mean TS:beta-actin gene ratio of 15.4 +/- 2.6 x 10(-3) (range, 2.7-35.9; P < 0.01). TS protein and TS mRNA expression are highly correlated, and each predict for response to 5-FU/LV-based chemotherapy in patients with colorectal and gastric cancer.

Actins↗
PubMed1994

Incorporation of 5-fluorouracil into U2 and U6 snRNA inhibits mRNA precursor splicing.

The splicing activities of 5-fluorouracil (FUra)-substituted U2 and U6 small nuclear RNAs (snRNAs) were examined in an in vitro splicing system. Yeast splicing extracts were specifically depleted of endogenous U2 and U6 snRNAs by antisense oligonucleotide-directed RNase H hydrolysis. Splicing activity was recovered when the extracts were reconstituted with synthetic U2 and U6 snRNAs. However, U2 snRNA with all uracils substituted with FUra (FU2) did not restore any splicing activity. Nondenaturing gel electrophoresis showed that FU2 failed to promote the assembly of spliceosome complexes. The ability of U2 snRNA to restore splicing in U2-depleted extracts increased as FUra content decreased but was still only 60% of control activity at 25% substitution of uracils with FUra. Addition of FU2 to nondepleted extracts caused strong inhibition of splicing accompanied by increased degradation of the pre-mRNA, suggesting that FU2 forms an inactive complex with a protein splicing factor that normally binds to the pre-mRNA. FU6 restored full splicing activity to U6-depleted extracts, but at a 5-fold higher concentration than U6 snRNA. These results demonstrate that the incorporation of FUra can impair the functions of catalytic RNA molecules.

Base Sequence↗
PubMed1994

Effect of 5-fluoro- and 5-bromouracil substitution on the translation of human thymidylate synthase mRNA.

The synthesis of thymidylate synthase (TS) from 5-fluorouracil (FUra)- and 5-bromouracil (BrUra)-substituted mRNAs was examined to investigate the effect of incorporation of uracil (Ura) analogs on translation. Human TS cDNA was transcribed in the presence of Ura-, FUra-, or BrUTP to obtain 100% substituted mRNA. The mRNAs were translated in a rabbit reticulocyte lysate system. The TS protein that was formed from each of the templates reacted identically with TS antibody in Western blots. Time courses of TS formation revealed a characteristic peak which occurred at 45 min for the Ura- and FUra-RNAs and at 2 h for the BrUra-RNA. Substitution of Ura with FUra did not alter the rate of translation, while substitution of BrU for Ura decreased the rate of translation. Substitution of Ura with FUra or BrUra enhanced the stability of the mRNAs in the rabbit reticulocyte lysate by 3- and 10-fold, respectively. Incorporation of BrUra influenced the binding and catalysis on the ribosome, resulting in a 3.5-fold greater rate of activation (Kact) and 6-fold lower Vmax than the equivalent values for the Ura- and FUra-substituted mRNAs. Nondenaturing gel electrophoresis revealed that different conformations exist among the mRNAs. These data show that translation can be influenced by the incorporation of fraudulent bases into mRNA and those bases that stabilize RNA secondary structure will have the greatest inhibitory effect on translation.

Base Sequence↗
PubMed1993

Differential S100 beta expression in choroidal and skin melanomas: quantitation by the polymerase chain reaction.

PURPOSE: S100 beta, a member of a calcium-binding protein family (S100s), is an important clinical marker for skin melanoma. In contrast, uveal melanomas appeared to express S100 beta protein less frequently and to a lesser degree. This study was performed to verify and extend this finding to the mRNA level. METHODS: A quantitative polymerase chain reaction (PCR)-based method was used. A ratio, comparing the S100 beta PCR fragment to that of beta-actin (an internal reference gene), was generated to compare S100 beta mRNA expression among samples. RESULTS: The ratios for skin melanomas (1.2 to 3.9; three tissues and two cell lines) were significantly higher than that for choroidal melanomas (0.1 to 0.63; seven of eight primary tumors and four of four cell lines). Only one choroidal melanoma biopsy had a ratio greater than 1. The PCR products from choroidal melanoma were identical in size and sequence to the S100 beta, as determined by gel electrophoresis and RNA conformational polymorphism. Because the ratios were also low in choroidal melanoma cell lines, the S100 beta phenotype appears to be genetically stable. CONCLUSION: S100 beta is differentially expressed at the RNA and protein levels by skin and choroidal melanomas, which are derived from distinct populations of melanocytes. However, choroidal melanomas expressing little or no S100 beta were significantly stained by antiserum specific for the S100 protein family. Taken together, these data suggest that choroidal melanocytes express another, perhaps even novel, S100 protein(s).

Actins↗
PubMed1992

NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity.

NAD(P)H:quinone oxidoreductase (DT-diaphorase; DTD) is an obligate two-electron reductase which may play a role in the bioactivation of antitumor quinones such as mitomycin C (MMC). We studied 10 colon carcinoma cell lines showing different levels of DTD activity (range, 0-3447 nmol/min/mg protein), as measured by the reduction of dichlorophenolindophenol. Expression of the NAD(P)H:quinone reductase gene (NQO1), which codes for the DTD enzyme, as measured by a polymerase chain reaction amplification technique was then correlated with enzymatic activity in all cell lines. HT-29 cells, which have intermediate DTD activity (769 +/- 144 nmol/min/mg protein, mean +/- SD) and are sensitive to MMC, showed high NQO1 expression relative to beta-actin (taken as 100% here for comparative purposes). BE cells which have no detectable DTD activity and are resistant to MMC showed moderate NQO1 expression (91% of HT-29). RNA single-strand conformational polymorphism analysis and subsequent sequencing of BE complementary DNA revealed a C to T mutation in the NQO1 complementary DNA. This confers a proline to serine substitution in the amino acid sequence of the protein. Additionally, HCT-116 cells showed both moderate DTD activity (390 +/- 41 nmol/min/mg protein) and NQO1 expression (41% of HT-29), while resistant subclones of these cells, exposed to MMC during 11 and 44 weeks, showed low gene expression (5 and 9% of HT-29 respectively) and enzymatic activity (11 +/- 6 and 36 +/- 16 nmol/min/mg protein). These results support the ideas that reductive activation of MMC by DTD may be important in the cytotoxicity of MMC and that polymerase chain reaction may be a useful technique for quantitating the relative expression of genes in human tumors.

Actins↗