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H J Lenz

Publications and source records attributed to H J Lenz.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

High basal level gene expression of thymidine phosphorylase (platelet-derived endothelial cell growth factor) in colorectal tumors is associated with nonresponse to 5-fluorouracil.

The gene expression levels of the nucleoside cleavage enzyme/angiogenic factor thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor, were measured by quantitative reverse transcription-PCR in 38 pretreatment biopsies of colorectal tumors from patients who were subsequently treated with 5-fluorouracil (5-FUra) and leucovorin (LV). The range of TP gene expression (relative mRNA levels) in those tumors nonresponsive to 5-FUra was much broader than that of the responding tumors. In contrast to in vitro studies that had shown that an increased intracellular level of TP potentiates the activity of 5-FUra by converting it to the more cytotoxic nucleoside form 5-fluoro-2'deoxyuridine, tumors with the highest basal TP expressions were nonresponders to 5-FUra/LV therapy. The mean TP mRNA level in the nonresponding tumors was 2.6-fold higher than that of the responding patients. We had shown previously that high expression of thymidylate synthase (TS), the target enzyme of 5-FUra, was also a predictor of nonresponse to 5-FUra (L. Leichman et al, J. Clin. Oncol., 15: 3223-3229, 1997). TP and TS expressions were found to be independent variables in these tumors, so that low expression levels of both TS and TP in tumors predicted a very high response rate (11 of 14) to 5-FUra/LV as well as a significantly longer survival, whereas none (0 of 24) of the patients with high expression of either TP or TS were responders.

Antimetabolites, Antineoplastic↗
PubMed1997

High frequency of simultaneous loss of p16 and p16beta gene expression in squamous cell carcinoma of the esophagus but not in adenocarcinoma of the esophagus or stomach.

Quantitative reverse transcription PCR (RT-PCR) was used to measure gene expressions (relative mRNA levels) of p16 and the alternate transcript pl6beta in esophageal and gastric tumors. p16 gene expression was undetectable in 13 of 25 esophageal squamous cell carcinomas. In 11 of these tumors, pl6beta was simultaneously missing whereas two of the pl6-deficient tumors still expressed p16beta. Among 34 esophageal adenocarcinomas and 11 gastric adenocarcinomas, only one tumor lacked p16 expression and all tumors expressed p16beta. p16 sequences were not detectable by PCR in genomic DNA from tumors lacking both p16 and p16beta mRNA, suggesting that the simultaneous loss of both gene expressions resulted from homozygous genomic deletion of the p16 gene. However, DNA from tumors that lacked p16 mRNA but expressed pl6beta did contain the p16 gene, consistent with loss of p16 expression in these tumors by transcriptional suppression. No point mutations in p16 cDNA were detected among 12 that were sequenced, but one p16 cDNA from a squamous cell carcinoma had a 19-base deletion, possibly indicating a splice-site mutation. Among those tumors that expressed p16 mRNA, the gene expression values of both p16 and pl6beta varied over a wide range. In some cases, p16 expression was detectable but low, suggesting that down-regulation of p16 expression may be used in some cases to achieve the funtional equivalent of gene deletion or transcriptional silencing. These results demonstrate that p16 expression patterns differ based on tumor histology and origin. Homozygous deletion of p16 appears to be common in esophageal squamous cell carcinomas but in adenocarcinomas, both gene deletion and transcriptional silencing of p16 were infrequent.

Adenocarcinoma↗
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↗
PubMed1997

Quantitation of intratumoral thymidylate synthase expression predicts for disseminated colorectal cancer response and resistance to protracted-infusion fluorouracil and weekly leucovorin.

PURPOSE: Response rates to fluorouracil (5-FU)-based therapy remain low. As new, active agents are being tested, information regarding specific intratumoral genetic determinants of chemotherapy sensitivity or resistance can be used to plan therapy rationally. Intratumoral thymidylate synthase (TS) quantitation may be among the most important determinants of sensitivity or resistance to 5-FU. MATERIALS AND METHODS: Forty-six disseminated colorectal cancer patients had measurable tumor biopsies for polymerase chain reaction (PCR)-based determination of TS mRNA pretreatment. Protracted infusion of 5-FU 200 mg/m2/d for 21 days with weekly intravenous leucovorin 20 mg/m2 each cycle was given. After two cycles, responses were evaluated. Response data were correlated with independently determined intratumoral ratios of TS/beta-actin mRNA for each patient. RESULTS: TS/beta-actin ratios were successfully obtained for 42 patients (91%). TS/beta-actin ratios ranged from 0.3 x 10(-3) to 18.2 x 10(-3) (median, 3.5 x 10[-3]). Twelve patients (26%) responded to treatment (median TS/beta-actin ratio, 1.7 x 10[+3]). Thirty-four patients did not respond (median TS/beta-actin ratio, 5.6 x 10[-3]). No patient with a TS mRNA level greater than 4.1 x 10(-3) responded. The median TS/beta-actin ratio (3.5 x 10[-3]) significantly segregated responders from nonresponders (P = .001). Median survival for patients with TS/beta-actin ratios < or = 3.5 x 10(-3) was 13.6 months; for patients with TS/beta-actin ratios greater than 3.5 x 10(-3), it was 8.2 months (P = .02). CONCLUSION: For this cohort, the intratumoral TS/beta-actin ratio had a statistically significant association with response and survival. This relationship for other 5-FU schedules remains unknown. Confirmation of these data in a larger patient population could lead to determination of therapy for disseminated colorectal cancer based on a specific intratumoral molecular parameter.

Actins↗
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↗
PubMed1995

Gene amplification and multidrug resistance induced by the phosphatase-inhibitory tumor promoter, okadaic acid.

The mechanism by which tumor promoters contribute to cellular transformation and tumorigenesis is not completely understood. To investigate further the molecular events involved in these processes, we used okadaic acid, a non-phorbol ester type tumor promoter that specifically inhibits certain protein phosphatases. We describe here that the continuous treatment of murine NIH 3T3 fibroblast cell cultures with okadaci acid resulted in a 50-fold amplification of two genes, mdr-1a and mdr-1b, that conferred multidrug resistance. As a consequence, the cells became cross-resistant to the cytotoxic effects of adriamycin, an antineoplastic drug used in the treatment of human tumors. Since genetic changes have been correlated with cell transformation and tumorigenesis, our results suggest that these processes may constitute an additional factor contributing to tumor promotion by okadaic acid.

3T3 Cells↗
PubMed1995

Effects of cerebral TRH on intestinal water transport: role of vagal, muscarinic, and VIP pathways.

Thyrotropin-releasing hormone (TRH) is a central nervous system (CNS) transmitter that stimulates various gastrointestinal secretory and motor processes by increasing vagal outflow. In this study, the CNS effects of TRH on ileal and jejunal water transport were examined in awake rats and dogs, respectively. Cerebral but not intravenous TRH (0.1-5.0 nmol/kg) significantly (P < 0.01) reversed net water absorption from approximately 30 microliters.cm-1.h-1 in rats and 300 microliters.cm-1.h-1 in dogs toward net water secretion of 60 and 600 microliters.cm-1.h-1, respectively. Truncal vagotomy and ganglionic blockade with chlorisondamine completely abolished this stimulatory effect of cerebral TRH, whereas adrenalectomy, hypophysectomy, noradrenergic and opiate blockade, and inhibition of prostaglandin and nitric oxide synthesis did not. Atropine methylnitrate significantly (P < 0.05) attenuated the stimulatory response produced by TRH by approximately 30%. Intravenous infusion of the vasoactive intestinal peptide (VIP) receptor antagonist, [4Cl-D-Phe6, Leu17]VIP (0.05-5.0 mumol.kg-1.h-1), significantly (P < 0.01) inhibited the stimulatory response of TRH by approximately 60%. Pretreatment of the animals with both atropine and the VIP antagonist completely abolished ileal and jejunal water secretion stimulated by cerebral TRH. These results indicate that 1) TRH acts within the CNS to stimulate net ileal and jejunal water secretion in rats and dogs, respectively; 2) these actions are mediated by vagal pathways; and 3) stimulation of intestinal secretion by cerebral TRH is primarily mediated by a VIP-sensitive mechanism and, in part, by a muscarinic mechanism.

Animals↗
PubMed1995

Transfection of wild-type but not mutant p53 induces early monocytic differentiation in HL60 cells and increases their sensitivity to stress.

HL60 cells, which lack the p53 gene due to a deletion, were used as an in vitro model system to study the effect of wild-type p53 gene expression on hematopoietic differentiation. We transfected HL60 cells with wild-type p53 and two mutant p53 cDNAs encoding the Val to Ala mutation at codon 143 and the Arg to Trp mutation at codon 248. Flow cytometry, growth, and cytochemical analysis for alpha-napthyl butyrate esterase activity and nitroblue tetrazolium reduction indicated that wild-type p53 but not mutant p53 induced early monocytic differentiation in the transfected HL60 cells without terminal growth arrest. The wild-type p53 transfectants did not differentiate along the granulocytic pathway, even when induced with 1.25% DMSO for 6 days; rather, these cells resembled monocytic cells, confirming that wild-type p53 transfection caused these cells to become committed to differentiate along the monocytic pathway. HL60 cells transfected with wild-type p53 were more sensitive to stress, such as growth in serum-depleted medium and exposure to a chemotherapeutic agent, etoposide.

Antigens, Surface↗
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↗
PubMed1994

Quantitative determination of the ratio of mutated to normal ras genes in the blood of leukemia patients by allele-specific PCR.

By combining allele-specific PCR amplification and a PCR-based quantitation approach, a method has been developed to estimate the mutated K-ras gene content in the blood of AML patients as a percentage of total K-ras. One PCR primer set was designed not to discriminate between mutant K-ras and wild-type K-ras and thus amplified the total K-ras gene. The other PCR primer set was designed to be allele-specific for K-ras gene containing a G to C mutation at codon 12. This primer set could discriminate the mutant and wild-type genes when the proportion of the mutated sequence was 0.2% of the total K-ras gene. To test the method on biological specimens, genomic DNA samples were analyzed from the peripheral blood of a patient who had secondary AML with the same codon 12 K-ras mutation. Two samples taken from this patient 2 months apart during follow-up had myeloblast cell contents of 67 and 80%. However, the percentage of mutated K-ras was 50% in both samples, suggesting that this patient may be inherently heterozygotic in this particular mutation. This ratio of mutated to normal K-ras in the patient's cells was confirmed by RNA-SSCP analysis and RNA sequencing. This quantitation method can provide a sensitive and specific estimation of the content of mutated K-ras alleles in patient samples.

Alleles↗
PubMed1994

Detection of clonal T-cell populations in gastrointestinal lymphomas by analysis of cRNA conformational polymorphisms of rearranged T-cell-receptor-gamma genes.

Analysis of complementary RNA molecules of junctional regions of rearranged T-cell-receptor-gamma genes show a pattern of conformational polymorphisms which is specific for an individual lymphocytic clone. In a blinded study we analysed formalin-fixed, paraffin-embedded histological specimens from gastrointestinal lymphomas and control tissues (lymphomas: pleomorphic T-cell 10, anaplastic large cell [Ki1+] 9, centroblastic 5, immunocytoma 1, B-CLL 2, Hodgkin's 2, centroblastic-centrocytic 1, MALT [mucosa associated lymphoid tissue] 1, T-cell acute lymphoblastic leukaemia 1, non-lymphoid or polyclonal lymphoid tissues 5). Junctional regions of rearranged TCR-gamma genes were amplified by the polymerase chain reaction and the products were transcribed into cRNA. Conformational patterns of cRNA molecules were analysed by polyacrylamide gel electrophoresis. 13/20 T-lineage lymphomas and the T-cell acute lymphocytic leukaemia displayed a distinct cRNA band pattern, all B-lineage lymphomas and the non-lymphoid control tissues were negative. Only one case of nasopharyngeal (lymphoepithelial, Schmincke-Regaud) carcinoma showed a faint cRNA banding pattern. This novel and non-radioactive assay allows for the rapid detection and molecular characterization of clonal lymphoid populations in minute histological biopsy specimens.

Base Sequence↗
PubMed1994

Quantitative analysis of folylpolyglutamate synthetase gene expression in tumor tissues by the polymerase chain reaction: marked variation of expression among leukemia patients.

Evidence from previous in vitro studies indicates that the enzyme folylpolyglutamate synthetase (FPGS) may be an important determinant of the antitumor activity of antifolate drugs that are substrates for this enzyme. To facilitate investigations regarding the association between FPGS content of tumor tissues and the sensitivity of tumors to antifolates, we developed a polymerase chain reaction (PCR)-based gene expression quantitation assay for measuring relative amounts of FPGS mRNA in tumor tissue specimens. From the known sequence of the human gene, FPGS-specific PCR primers were chosen that flanked a 263-base segment of the FPGS gene. The PCR carried out with these primers was linear over at least a three orders of magnitude range of starting cDNA concentration. The amount of cDNA required per assay corresponded to the quantity of RNA contained in nanogram to microgram amounts of tissue, depending on the level of gene expression. In CHO AUXB1 (FPGS) cell lines transfected with human DNA and expressing different levels of human FPGS, FPGS gene expression measured by this assay was linear with the FPGS enzyme activity in the cells. In human head and neck cell lines, which contained naturally varying levels of FPGS enzyme activity, FPGS gene expressions were also linearly proportional to FPGS enzyme content as measured both by activity in cell-free extracts and by intracellular methotrexate polyglutamate formation. Among leukemic cells from 11 acute lymphocytic leukemia and acute myelogenous leukemia patients, FPGS expression varied by over 500-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗