Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.     

Transgenic tomato plants expressing recA and NLS-recA-licBM3 genes as a model for studying meiotic recombination

Homologous DNA recombination in eukaryotes is necessary to maintain genome stability and integrity and for correct chromosome segregation and formation of new haplotypes in meiosis. At the same time, genetic determination and nonrandomness of meiotic recombination restrict the introgression of genes and generation of unique genotypes. As one of the approaches to study and induce meiotic recombination in plants, it is recommended to use the recA gene of Escherichia coli. It is shown that the recA and NLS-recA-licBM3 genes have maternal inheritance and are expressed in the progeny of transgenic tomato plants. Plants expressing recA or NLS-recA-licBM3 and containing one T-DNA insertion do not differ in pollen fertility from original nontransgenic forms and can therefore be used for comparative studies of the effect of bacterial recombinases on meiotic recombination between linked genes.     

Analysis of the meiotic recombination frequency in transgenic tomato hybrids expressing <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">NLS-recA-licBM3</Emphasis> genes

To study and induce meiotic recombination in plants, we generated and analyzed transgenic tomato hybrids F₁-RecA and F₁-NLS-recA-LicBM3 expressing, respectively, the recA gene of Escherichia coli and the NLS-recA-licBM3 gene. It was found that the recA and NLS-recA-licBM3 genes are inherited through the maternal and paternal lineages, they have no selective influence on the pollen and are contained in tomato F₁-RecA and F₁-NLS-RecA-LicBM3 hybrids outside the second chromosome in the hemizygous state. The comparative analysis of the meiotic recombination frequency (rf) in the progenies of the transgenic and nontransgenic hybrids showed that only the expression of the recA gene of E. coli in cells of the F1-RecA plants produced a 1.2–1.5-fold increase in the frequency of recombination between some linked marker genes of the second chromosome of tomato.     

Functional activity of ABC transporters (markers of multidrug resistance) in human colon adenocarcinoma and normal colonic mucosa]

Functional activity of multidrug resistance (MDR) markers (total activity of ABC-transporters, P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) activities) in human colon adenocarcinoma and normal mucosa was examined. Functional activity of ABC-transporters was revealed in all colon tumors and in 70% of normal mucosa samples investigated. Expression of Pgp and MRP functional activity was determined in about 50% and 70% of colon tumors respectively. Pgp+MRP+ phenotype was determined in 36% of normal mucosa and adenocarcinoma samples. Expression of Pgp+MRP- phenotype was practically the same in normal mucosa and tumors (in 10 and 18% of samples respectively). Pgp-MRP+ phenotype was revealed two times more often in tumors than in mucosa--in 36 and 18% respectively. On the contrary, Pgp-MRP- phenotype was detected more rarely in tumors than in mucosa (in 10 and 36% of samples respectively). Transporters different from Pgp and MRP were also determined in some tumors and normal mucosa. At the patients with expression of Pgp function in normal mucosa the activity of the transporter was revealed in 25% of tumor samples only. On the contrary, at the patients with expression of MRP function in normal mucosa the activity of the transporter was revealed in 70% of tumor samples. At the patients with no expression of Pgp or MRP activity in normal mucosa the function of the transporters in tumors was determined in 60% and 70% of samples respectively. It is concluded that functional activity of various ABC-transporters (Pgp, MRP and other different from Pgp and MRP) is expressed in human colon adenocarcinoma; expression of ABC-transporters functional activity in normal mucosa does not predict MDR phenotype of the tumor.     

Systemic polychemotherapy in combined treatment of colon cancer]

Seven hundreds and fifty three patients with colon cancer were operated in the Unit of Colon Surgery of the State Scientific Centre of Coloproctology within the period from 1998 to 2003. 352 (46.7%) of them were subjected to systemic chemotherapy. The patients subjected to the surgical and antitumor drug therapy were divided into 3 groups according to the main disease stage: 103 (29.3%) patients with the disease stage T4N0M0 (including those with the tumor local spread), 116 (32.9%) patients with the disease stage T(3-4)N+M0 and 133 (37.8%) patients with the disease stage T(3-4)NxM1. In the patients with the colon serous membrane diffusion and locally spread tumors (total of 103 patients) 3- and 5-year survival was stated in 81.0 +/- 2.8% and 76.5 +/- 3.7% respectively. The control group included 85 patients subjected to the surgical treatment alone. In the latter group 5-year survival was recorded in 68.0 +/- 2.9% of the patients. In the group of the patients with affection of the regional lymph nodes subjected to antitumor drug therapy 3- and 5-year survival was recorded in 64.1 +/- 3.5% and 57.5 +/- 4.2% respectively. In 52 patients not subjected to the systemic chemotherapy the analogous index was equal to 44.5 +/- 3.4%. The systemic chemotherapy was applied to 111 (78.2%) patients with metastases to the liver. In 50 of them cytoreductive operations were performed and in 61 patients palliative resection of the colon was carried out. The average period without clinical manifestations of the disease in the patients subjected to the colon palliative resection and systemic chemotherapy was equal to 8.5 +/- 3.5 months. In the patients with metastases to the liver subjected to the cytoreductive operations the average lifespan after the operations amounted to 24.5 months. The average lifespan of the patients with canceromatosis subjected to the systemic chemotherapy and cytoreductive operations amounted to 13.5 months. When the chemotherapy in such patients was effective the average lifespan was 16 months, while in case of the therapy failure it was 8.5 months.     

Cytoreductive surgery with intraperitoneal chemotherapy in patients with colon cancer and peritoneal carcinomatosis]

The efficacy of cytoreductive approach for colon cancer with carcinomatosis was assessed. 34 patients (the main group) underwent colon resections, peritonectomy, omentectomy and intraperitoneal chemotherapy (mitomycin and 5-fluorouracil). 22 patients (control group) underwent palliative colon resections only. The spread of peritoneal dissemination was assessed in all patients, basing on Peritoneal Cancer Index (PCI). It was found, that the rate of postoperative morbidity is higher in the main group, but it was not accompanied by enhanced postoperative mortality. It was also detected, that cytoreductive approach permits to prolong the survival (17.0 +/- 3.5 months in main group vs. 7.5 +/- 2.1 months in controls). The main prognostic factor in these patients was the PCI. The most favorable prognosis is identified if PCI < 5 (few discrete implants are present); the survival in this group was 23.0 +/- 4.5 months and disease-free interval was 14.0 +/- 2.5 months. When PCI was higher then 5, the survival was 14.5 +/- 2.5 months and disease-free interval was 7.0 +/- 1.3 months.     

Combined management of colon cancer local recurrences with using intraabdominal chemotherapy

The efficacy of intraabdominal chemotherapy in management of local recurrences of colon cancer was studied. Intraabdominal intraoperative and early postoperative chemotherapy made it possible to prolong of the recurrenceless term and to improve the life quality of the patients with local recurrences of colon cancer. There was observed a tendency to increase the five-year survival.