Biomedical subjects

M Kalipciyan

Publications and source records attributed to M Kalipciyan.

3 recordsLinked to original sources

PubMed2001

Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: artificial generation of deletions in ribonucleotide reductase mRNA.

Using reverse transcriptase-polymerase chain reaction (RT-PCR), we have recently described a bona fide deletion within the coding sequence of the large subunit of ribonucleotide reductase (R1) mRNA in colon cancer. Consecutive studies have raised questions about the nature of this phenomenon, because the corresponding genomic alteration at the DNA level or an aberrant protein could not be detected. Thus we considered an in vitro artifact during RT-PCR as a possible explanation for this observation. In contrast to reverse transcriptase, Taq DNA polymerase or C. therm DNA polymerase did not generate the aberrant product, suggesting the demand for the template switching activity intrinsic to retroviral reverse transcriptases. In fact, virtually the same deletion was observed in RT-PCR experiments when in vitro transcribed R1 mRNA was used. Considering structural prerequisites for template switching within R1 mRNA, we show that two direct repeats adjacent to a strong stem-loop secondary structure flank the deleted region of 1851 base pairs. Because several mRNAs encoding proteins of clinical and diagnostic importance fulfill these criteria, template switching enhances the potential risk of observing artifacts when interpreting results from RT-PCR studies. As shown in the present example, this may involve the artificial generation and the misinterpretation of PCR fragments amplified from targets relevant to tumor biology or cancer pharmacology. As a possible solution, one-step PCR with C. therm polymerase should be considered. This polymerase eliminates the artificial generation of aberrant mRNA signals observed during cDNA synthesis.

Adenocarcinoma↗

PubMed2000

Genetic analysis of multiple synchronous lesions of the colon adenoma-carcinoma sequence.

The colorectal adenoma-carcinoma sequence represents a well-known paradigm for the sequential development of cancer driven by the accumulation of genomic defects. Although the colorectal adenoma-carcinoma sequence is well investigated, studies about tumours of different dignity co-existent in the same patient are seldom. In order to address the distribution of genetic alterations in different lesions of the same patient, we coincidently investigated carcinomas, adenomas and aberrant crypt foci in patients with sporadic colon cancer. By utilizing polymerase chain reaction, single-strand conformation polymorphism, heteroduplex-analysis, restriction fragment length polymorphism, protein truncation test and sequencing techniques we looked for mutations and microsatellite instability of APC, H-ras, K-ras, p53, DCC and the DNA repair genes hMLH1/hMSH2. In accordance with the suggested adenoma-carcinoma sequence of the colon, four patients reflected the progressive accumulation of genetic defects in synchronously appearing tumours during carcinogenesis. However, two patients with non-hereditary malignomas presented different genetic instabilities in different but synchronously appearing tumours suggesting non-clonal growth under almost identical conditions of the environment. Thus, sporadically manifesting multiple lesions of the colon were not necessarily driven by similar genetic mechanisms. Premalignant lesions may transform into malignant tumours starting from different types of genetic instability, which indicates independent and simultaneous tumorigenesis within the same organ.

Adenocarcinoma↗

PubMed1997

Transcription and activity of 5-fluorouracil converting enzymes in fluoropyrimidine resistance in colon cancer in vitro.

Cellular resistance to 5-fluorouracil (5-FU) is not completely understood. Since 5-FU shares the pyrimidine pathway with the physiological pyrimidines, we investigated the relationship between fluoropyrimidine metabolism, nucleic acid uptake and cytotoxicity of 5-FU in eight colon tumour cell lines including 5-FU-resistant subclones. The cytotoxicity of 5-FU was increased up to 423-fold when the anabolites 5-fluorouridine (FUrd), 5-fluorodeoxyuridine (FdUrd), and 5-fluorodeoxyuridine monophosphate (FdUMP) were compared with the parent drug in vitro. The enzymes uridine phosphorylase and thymidine phosphorylase were predictive for the cytotoxicity of 5-FU in 5/7 cell lines. Inhibition of uridine phosphorylase and thymidine phosphorylase by antisense strategies effectively antagonised 5-FU, abolishing 84% and 79% of its toxicity. The importance of thymidine phosphorylase was supported by a highly restricted enzyme activity in 5-FU-resistant cells. In 5-FU naive cells, a stimulating effect of 5-FU on thymidylate synthase mRNA and ribonucleotide reductase mRNA expression was observed. In these cells, antisense oligonucleotides to ribonucleotide reductase significantly reduced cell growth. Downregulation of ribonucleotide reductase mRNA in 5-FU-resistant subclones suggests different mechanisms in primary and secondary resistance to 5-FU. Most of the intracellular 5-FU was selectively incorporated into RNA (range: 45-91%) and generally spared DNA (range: 0.2-11%). In synthesising our data, we conclude that drug resistance could be overwhelmed through bypassing limiting steps in the activation of 5-FU. In the majority of colonic tumours, the activity of uridine phosphorylase and thymidine phosphorylase may have prognostic relevance for the cytotoxicity of 5-FU in vitro.

Antimetabolites, Antineoplastic↗