Nikolai Mironov

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.

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.

L-myc allele polymorphism and prognosis for metastases in Russian gastric cancer patients.

During recent years, several attempts have been made to use restriction fragment length polymorphism (RFLP) of protooncogenes, e.g. c-Ha-rus and L-myc, as a marker for prognosis in human cancers [I]. L-myc has L and S alleles, and the presence of two S-S alleles has been reported to be associated with metastases of lung [2] and stomach cancer [3] in Japanese patients, although another study of lung cancer in Caucasians showed no such association [4]. Stomach cancer is common among the Russian population and a good prognostic marker is greatly needed. We performed L-myc RFLP analysis of stomach cancer samples from 21 newly diagnosed patients in Russia. DNA from tumour samples and corresponding mucosa was amplified by polymerase chain reaction, before single strand conformation polymorphism analysis or EcoRI digestion [4]. 9 patients had metastases in regional lymphnodes. All the tumours contained the same L-myc genotypes as the corresponding normal mucosa. Among the 21 patients, the ratio of L-myc genotypes between L-L, L-S and S-S was 0.24:0.62:0.14, which is very close to that found for a healthy white Caucasian population [4]: 0.25:0.62:0.13 (P=O.9865, x2). Among the nine patients with metastases, only 1 patient was found to have the SS genotype, anddistribution ofL-mycalleles was 0.33:0.56:0.11. The distribution of alleles in patients without metastases was 0.17:0.66:0.17, which did not significantly differ from the patients with metastases (P=O.8137, x2). Thus, no excess prevalence of S-S L-myc allele genotype was found in these Russian patients with metastases. If the reported association between the S-S genotype and metastases in Japanese patients could be confirmed in future studies, such an association might be explained by the presence of some specific factor(s) (internal or external) modulating the metastatic process. Among such factors could be ethnic differences in genotypes in the L-myc gene and environmental risk factors. However, because loss of heterozygosity is infrequent in stomach cancer at the L-myc locus [S], the loss of putative metastasis suppressor gene near this locus is improbable. In addition, it appears that the L-myc gene itself is

Alkylation Repair in Human Tissues

The first evidence of alkylation of DNA by environmental chemicals was provided by Brookes and Lawley (1960, 1961) who showed that alkylating mustard gas formed 7-alkylguanine in nucleic acids; mustard gas was reported in 1946 (Auerbach and Robson) to be a chemical mutagen and respiratory tract tumours were observed among workers involved in its manufacture (Wada et al., 1968). The carcinogenicity of the alkylating nitrosamine dimethylnitrosamine (DMN) was first identified in the mid 1950s (Magee and Barnes, 1956) and in the subsequent decade the importance of DNA alkylation in the mutagenicity and carcinogenicity of such alkylating agents became apparent. The development of the thoughts underlying research on carcinogenic alkylating agents have been recently described in an interesting review by Lawley (1989).

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.

Measurement of the removal of O6-methylguanine and O4-methylthymine from oligodeoxynucleotides using an immunoprecipitation technique

A sensitive and rapid procedure for measurement of alkyltransferase repair activity involving oligodeoxynucleotides followed by immunoprecipitation is described. Dodecadeoxynucleotides containing O6-methylguanine or O4-methylthymine were used as substrates for alkyltransferases and the reaction products of methylated or demethylated substrates were separated by precipitation with highly specific antibodies. This approach for O6-alkylguanine-DNA alkyltransferase measurement is far more rapid than when the reaction products are separated by chromatography. This technique makes the assay applicable to large-scale epidemiological or clinical studies and suggests a similar methodology could be applied for other DNA repair enzymes.

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 nontumorigenic 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 μg/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. Mol. Carcinog. 23:20–24, 1998.