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Biomedical subjects

N Mironov

Publications and source records attributed to N Mironov.

16 recordsLinked to original sources
PubMed2001

Second primary glioblastoma.

Although characterized by a highly variable phenotype and multiple genetic alterations, glioblastomas are considered monoclonal in origin. We here report on a 64-yr-old patient who developed a second glioblastoma in the left frontal lobe 10 yr after surgical resection of a glioblastoma of right frontal lobe. The first tumor contained 2 p53 mutations, in codon 213 (CGA-->TGA, Arg-->stop) and codon 306 (CGA-->TGA, Arg-->stop), further, 1 missense PTEN mutation (codon 257, TTC-->TTA, Phe-->Leu) and a silent PTEN mutation (codon 154, TTC-->TTT, Phe-->Phe). The second glioblastoma also contained multiple, but different mutations: p53 mutations in codons 158 (CGC-->CAC, Arg-->His) and 273 (CGT-->TGT, Arg-->Cys), and a PTEN mutation in codon 233 (CGA-->TGA, Arg-->Stop). Both neoplasms had a homozygous p16 deletion. The discordant pattern of mutations indicates that the second glioblastoma was not a recurrence but an independent second glioblastoma. The presence in these neoplasms of multiple mutations in tumor suppressor genes suggests the involvement of a novel disease mechanism but there was no indication of a DNA mismatch repair deficiency or of an inherited tumor syndrome.

Chromosomes, Human, Pair 10↗
PubMed2000

Genomic instability in multistage carcinogenesis.

For a normal cell to accumulate multiple genetic changes during multistage carcinogenesis, the induction of genomic instability is considered advantageous. Since most human cancers are associated with exposure to environmental carcinogens, it is likely that environmental carcinogens interact with genomic instability. Our results indeed suggest that carcinogens contribute to the induction of microsatellite instability and induce more mutations in those cells which show microsatellite instability. We have recently developed a sensitive method to clearly detect changes in simple repeats of coding sequences of cancer genes and the results suggest that such sequences of different genes are mutated in different tumors.

Animals↗
PubMed1999

Connexins in tumour suppression and cancer therapy.

Malignant cells usually show altered gap junctional intercellular communication and are often associated with aberrant expression or localization of connexins. Transfection of connexin genes into tumorigenic cells restores normal cell growth, suggesting that connexins form a family of tumour suppressor genes. Some studies have also shown that specific connexins may be necessary to control growth of specific cell types. Although we have found that genes encoding connexin32 (Cx32; beta 1), Cx37 (alpha 4) and Cx43 (alpha 1) are rarely mutated in tumours, our recent studies suggest that methylation of the connexin gene promoter may be a mechanism by which connexin gene expression is down-regulated in certain tumors. We have produced various dominant negative mutants of the genes encoding Cx26 (beta 2), Cx32 and Cx43, some of which prevent the growth control exerted by the corresponding wild-type genes. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through gap junctions. Recently, we and others have shown that gap junctional intercellular communication is responsible for the bystander effect seen in herpes simplex virus thymidine kinase/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.

Animals↗
PubMed1999

Genetic and epigenetic changes of intercellular communication genes during multistage carcinogenesis.

During multistage carcinogenesis, the functions of several key genes involved in cell growth control must be damaged. Such genes include not only those involved in cell cycle control of individual cells, but also those involved in the coordination of cell growth throughout a given tissue through cell-cell communication. The most intimate form of intercellular communication is mediated by gap junctions. Gap junctional intercellular communication (GJIC) is known to transfer small water soluble molecules, including cAMP and IP3, from the cytoplasm of one cell to that of its neighbors; the growth of a given GJIC-associated cell is thus kept in check by other GJIC-connected cells. Most tumor cells have a reduced ability to communicate among themselves and/or with surrounding normal cells, confirming the importance of intact GJIC in growth control. When connexin (gap junction protein) genes are transfected into such cells, normal cell growth control is often recovered. Certain dominant-negative mutant connexin genes can reverse such tumor suppression. While these results suggest that connexin genes form a family of tumor suppressor genes, so far we have found no connexin gene mutations in human tumors; only two connexin gene mutations were found in chemically induced rat tumors. On the other hand, our recent studies suggest that connexin genes may be inactivated by hypermethylation of their promoter regions, suggesting that epigenetic inactivation of connexin genes may be a mechanism of GJIC disturbance in certain tumors. However, in many tumor cells connexins are normally expressed but aberrantly localized. The mechanisms of aberrant localization of connexins include lack of an appropriate cell-cell recognition apparatus and aberrant phosphorylation of connexins. These results suggest that GJIC disorders may occur not only because of aberrant expression of connexin genes themselves, but also as a result of disruption of various control mechanisms of the protein functions.

Animals↗
PubMed1999

A novel sensitive method to detect frameshift mutations in exonic repeat sequences of cancer-related genes.

We have investigated frameshift mutations in exonic repeats in the ATR, BRCA1, BRCA2, PTCH, CTCF, Cx26, NuMa and TGFbetaRII genes, using human tumor samples from stomach, esophagus, breast and skin and melanoma, as well as colon cancer and endometrial cancer cell lines (125 samples in total). We developed a sensitive method to detect mutations in the repeats, using the introduction of an artificial restriction site into a repeat. The method detects a single mutant among 10(3) normal genes. Thus, an alteration in a repeated sequence can be detected unambiguously. The (A)(8) repeat of BRCA2 was found mutated in only two of five colon cell lines with microsatellite instability (MI(+)). The ATR gene has an (A)(10) repeat which was altered in two of three MI(+) stomach cancer samples and one of three MI(+) endometrial cell lines. The TGFbetaRII gene [with an (A)(10) repeat] had the maximal frequency of mutations: 10 out of 13 MI(+) samples. At least one sample from all types of cancers, except melanomas, was positive for TGFbetaRII gene mutations. No mutations were found in repeats in the BRCA1, PTCH, CTCF, NuMA and Cx26 genes in any types of tumors examined. In conclusion, our study indicates that repeats were altered only in MI(+) cells and that the mutation frequencies in the genes studied differ among tumor types. Based on these results, we discuss meaningful and meaningless alterations in exonic repeats.

Base Sequence↗
PubMed1998

Frequency of HPRT gene mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine corresponds to replication error phenotypes of cell lines.

We have examined whether cells with replication error-positive (RER+) and -negative phenotype (RER ) respond differently to the mutagen MNNG, employing three RER+ and two RER- human cell lines. Cells were treated with several concentrations of MNNG, and HPRT mutants were selected phenotypically by their growth in the presence of 6-thioguanine. While the variation of the mutation frequency within each group was about an order of magnitude, it was found that MNNG induced a level of mutations in the HPRT gene some 100- to 1000-fold higher in RER+ cells than in cells with RER-phenotype. MNNG, at a concentration of 30 microM, produced a mutation frequency 450-fold higher in HCT116 (RER+) cells than in SW480 (RER-) cells. Our findings suggest that the RER+ phenotype predisposes cells to MNNG-induced hypermutability.

Cell Line↗
PubMed1998

Malignant transformation of simian virus 40-immortalized human milk epithelial cells by chemical carcinogenesis accompanied by loss of heterozygosity on chromosome 1 but not microsatellite instability.

Simian virus 40-immortalized human milk epithelial cells (HuMI) are anchorage dependent and non-tumorigenic but can spontaneously progress to anchorage-independent and tumorigenic cells. To see whether HuMI cells can be transformed into anchorage-independent cells by chemical carcinogens, we treated them with 3-methylcholanthrene (MCA, 10 microg/mL). After 7-8 wk of culture, none of the treated cells grew in soft agar. However, when HuMI cells treated with MCA were cultured with 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 ng/mL), they grew in soft agar; cells treated with TPA alone did not. TPA at this dose was cytotoxic to HuMI cells but not to their tumorigenic subline HuMI-TTu2. The response of the anchorage-independent HuMI-T cells was intermediate. These results indicate that HuMI cells can be transformed by treatment with MCA plus TPA, possibly because TPA selects those cells that are progressing toward malignancy. All five clones from MCA plus TPA-induced transformed cells formed malignant carcinomas in nude mice. When microsatellite changes at 17 loci in HuMI, HuMI-T, HuMI-TTu2, and five MCA plus TPA-transformed cells were examined, none of these cell lines showed instability at any locus, and no change in microsatellite length was found. However, all five MCA plus TPA-transformed cell lines showed loss of heterozigosity at 1q21-23 and 1q42 loci. This region of chromosome 1 is known to contain at least one antiproliferative gene, and our results suggest that inactivation of such a gene may be essential for full transformation of HuMI cells by chemical carcinogens.

Breast↗
PubMed1997

Increased genetic stability of HeLa cells after connexin 43 gene transfection.

To test the hypothesis that intact gap-junctional intercellular communication (GJIC) is necessary for genomic stability, we compared the spontaneous and chemically induced mutation frequencies in GJIC-proficient and -deficient HeLa cells. Thus, we determined microsatellite instability and mutation frequency in the HPRT gene in parental HeLa cells, which have no GJIC ability, and in HeLa cells in which GJIC was restored by transfection with the connexin 43 (Cx43) gene. When HeLa cells with (Cx43+) or without Cx43 gene (Cx43-) were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or methylnitrosourea, the Cx43+ cells survived better than Cx43- cells. The mutation frequency at CA repeats was measured with a shuttle vector; in the vector, the coding region of the beta-galactosidase gene was rendered out of frame by insertion of CA repeats, and the frame could be restored by insertion or deletion mutations of the CA repeats. The mutation frequency at CA repeats was 2-fold lower in Cx43+ cells than in Cx43-, both before and after exposure to MNNG or methylnitrosourea (P < 0.05). The frequency of spontaneous HPRT gene mutations, selected by their resistance to 6-thioguanine, was 3-fold lower in Cx43+ cells than Cx43- cells. Similarly, the frequency of MNNG-induced HPRT mutations was significantly higher in Cx43- cells (P < 0.001). Similar results were obtained even when the mutant selection process was carried out in the presence of alpha-glycyrrhetinic acid, a long-term inhibitor of GJIC, suggesting that the observed effect is not due to unwanted killing of cells by GJIC-mediated metabolic cooperation. Thus, our data demonstrate that HeLa cells transfected with the Cx43 gene become more resistant to spontaneous as well as chemically induced genetic changes.

Antimetabolites, Antineoplastic↗
PubMed1996

Human connexin 37 is polymorphic but not mutated in tumours.

Connexins are phylogenetically conserved proteins responsible for gap junctional intercellular communication (GJIC). In tumours, GJIC is frequently disrupted. We have tested the hypothesis that the connexin 37 (Cx37) gene might be mutated in human tumours from tissues in which the Cx37 gene is known to be expressed. Eight lung adenocarcinomas and 18 sporadic breast carcinomas were analysed. While most tumours had GTA at codon 130, a base change GTA-->ATA converting valine into isoleucine was found in three breast cancers (one homozygous for ATA) and two lung tumour samples. However, screening of normal DNA from the same patients and DNA from 42 healthy donors revealed that such base change also exists in normal tissue. Thus, we conclude that there is polymorphism of the connexin 37 gene in the human population. This is the first finding of polymorphism in the connexin gene family.

Adenocarcinoma↗
PubMed1996

Cx32 gene mutation in a chemically induced rat liver tumour.

Cx32 is a major gap junction protein of the liver and is often aberrantly expressed in liver tumours. We have studied mutation of the Cx32 gene during chemically induced hepatocarcinogenesis. DNA from 12 rat liver tumours induced by diethylnitrosamine or N-ethyl-N-hydroxyethylnitrosamine (EHEN) was analysed by the PCR/SSCP method. One tumour induced by EHEN harboured a G--> A transition mutation at codon 220, substituting His for Arg. When the mutant DNA was transfected into HeLa cells, which are deficient in gap junctional intercellular communication (GJIC), GJIC recovered, as in HeLa cells transfected with the wild-type Cx32 gene. Moreover, GJIC was modulated by cAMP, 12-O-tetradecanoylphorbol-13-acetate and lysophosphatidic acid similarly in mutant and wild-type Cx32 transfectants. These results suggest that Cx32 gene mutations are rarely involved in rat hepatocarcinogenesis and that the mutation found in a tumour may be functionally silent.

Adenoma↗
PubMed1995

Cell-type-specific ras mutations but no microsatellite instability in chemically induced mouse skin tumors and transformed 3T3 cells.

In mouse skin, both papillomas/carcinomas or fibrosarcomas can be induced by 7,12-dimethylbenz[alpha]anthracene (DMBA) depending on the mode of administration. Thus, upon DMBA painting (or transplacental exposure by i.p. injection to pregnant mothers) followed by 12-O-tetradecanoylphorbol-13-acetate applications to the skin of CD1 mice, papillomas and carcinomas appeared, whereas fibrosarcomas were induced when DMBA was s.c. injected. Molecular analysis of these tumors revealed that the majority of papillomas (17/20) and carcinomas (9/10) showed DMBA-specific mutations (A to T transversion at the 61st codon) in the Ha-ras gene. On the other hand, many fibrosarcomas (5/9) showed the same mutation only in the Ki-ras gene. When microsatellites were studied in these tumors at nine loci containing CA repeats, none of them showed an instability. In addition, when we analyzed 14 BALB/c 3T3 cell lines transformed by various carcinogens (including 3 clones induced by DMBA which have the A to T mutation in the Ki-ras gene), no changes in CA repeats were observed. These results suggest that DMBA-induced mouse tumors/transformed cells show cell-type-specific ras gene mutations, and these occur independently in the absence of microsatellite instability. While murine cells are considered to be relatively susceptible to cancer induction partially due to genomic instability, our results indicate that microsatellite instability is not induced in these cells by chemical carcinogens.

3T3 Cells↗
PubMed1995

Intercellular communication and carcinogenesis.

Two types of intercellular communication (humoral and cell contact-mediated) are involved in control of cellular function in multicellular organisms, both of them mediated by membrane-embedded proteins. Involvement of aberrant humoral communication in carcinogenesis has been well documented and genes coding for some growth factors and their receptors have been classified as oncogenes. More recently, cell contact-mediated communication has been found to have an important role in carcinogenesis, and some genes coding for proteins involved in this type of communication appear to form a family of tumor-suppressor genes. Both homologous (among normal or (pre-)cancerous cells) as well as heterologous (between normal and (pre)cancerous cells) communications appear to play important roles in cell growth control. Gap junctional intercellular communication (GJIC) is the only means by which multicellular organisms can exchange low molecular weight signals directly from within one cell to the interior of neighboring cells. GJIC is altered by many tumor-promoting agents and in many human and rodent tumors. We have recently shown that liver tumor-promoting agents inhibit GJIC in the rat liver in vivo. Molecular mechanisms which could lead to aberrant GJIC include: (1) mutation of connexin genes; (2) reduced and/or aberrant expression of connexin mRNA; (3) aberrant localization of connexin proteins, i.e., intracytoplasmic rather than in the cytoplasmic membrane; and (4) modulation of connexin functions by other proteins, such as those involved in extracellular matrix and cell adhesion. Whilst mutations of the cx 32 gene appear to be rare in tumors, cx 37 gene mutations have been reported in a mouse lung tumor cell line. Our results suggest that aberrant connexin localization is rather common in cancer cells and that possible molecular mechanisms include aberrant phosphorylation of connexin proteins and lack of cell adhesion molecules. Studies on transfection of connexin genes into tumor cells suggest that certain connexin genes (e.g., cx 26, cx 43 and cx 32) act as tumor-suppressor genes.

Animals↗
PubMed1994

Altered homologous and heterologous gap-junctional intercellular communication in primary human liver tumors associated with aberrant protein localization but not gene mutation of connexin 32.

Gap-junctional intercellular communication (GJIC) in 20 primary human liver tumors with different degrees of malignancy has been studied at the functional and molecular levels. When GJIC capacity was determined by dye-transfer assay performed directly with freshly removed tumor tissue, significant reduction was found in all samples, regardless of their morphology. In addition, a selective lack of GJIC between tumor and surrounding non-tumorous cells was observed in some cases, probably due to the physical separation between them resulting from encapsulation of tumors. There was, however, no essential change in the level of expression of the major liver gap-junction protein, connexin (cx) 32, in liver tumors as measured by Northern and Western blot analyses. Immunohistochemical study revealed aberrant localization of cx 32 in the majority of malignant liver tumors. Instead of cytoplasmic membrane localization at intercellular contacts, cx 32 was detected mainly either intracytoplasmically or in plasma membrane free from contact with other cells. We did not detect any mutation in the coding sequence of the cx 32 gene from any of the human liver tumors we tested. Thus it is likely that the aberrant localization of cx 32 in tumor cells is due to disruption of the mechanisms for establishment of this protein into gap-junction plaques, rather than to structural abnormality of the cx 32 protein itself. Another member of the connexin family, cx 43, not detectable in non-tumorigenic hepatocytes, was expressed in several tumors, especially in invasive areas, but was detected in only a few tumor cells and was localized intracytoplasmically, suggesting that cx 43 protein is not involved in GJIC in the tumors.

Adult↗