Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage.

The carboxyterminal cytoplasmic portions (tails) of desmosomal cadherins of both the desmoglein (Dsg) and desmocollin type are integral components of the desmosomal plaque and are involved in desmosome assembly and the anchorage of intermediate-sized filaments. When additional Dsg tails were introduced by cDNA transfection into cultured human epithelial cells, in the form of chimeras with the aminoterminal membrane insertion domain of rat connexin32 (Co32), the resulting stably transfected cells showed a dominant-negative defect specific for desmosomal junctions: despite the continual presence of all desmosomal proteins, the endogenous desmosomes disappeared and the formation of Co32-Dsg chimeric gap junctions was inhibited. Using cell transfection in combination with immunoprecipitation techniques, we have examined a series of deletion mutants of the Dsg1 tail in Co32-Dsg chimeras. We show that upon removal of the last 262 amino acids the truncated Dsg tail still effects the binding of plakoglobin but not of detectable amounts of any catenin and induces the dominant-negative phenotype. However, further truncation or excision of the next 41 amino acids, which correspond to the highly conserved carboxyterminus of the C-domain in other cadherins, abolishes plakoglobin binding and allows desmosomes to reform. Therefore, we conclude that this short segment provides a plakoglobin-binding site and is important for plaque assembly and the specific anchorage of either actin filaments in adherens junctions or IFs in desmosomes.

[Clinical significance of carriage of rare variants of connexin-26 genetic polymorphism in gastric cancer].

The authors present first results of investigations of the connexin-26 gene in DNA obtained from peripheral blood of 55 patients operated on for gastric cancer. Gastric cancer patients were found to have carriage of the Cx 26 gene that was reliably associated with the invasive ability of the tumor. Change of the connexin-26 gene in gastric cancer is evidence of an important role of intercellular gap junctions in the arising and development of gastric cancer.

Negative growth control of HeLa cells by connexin genes: connexin species specificity.

In order to examine whether different connexin gene species exert different degrees of tumor-suppressing activity, we characterized growth characteristics of a gap junction-deficient human cancer cell line, HeLa cells, before and after transfection with cDNA for three different connexins, connexin (cx) 26, cx 40, and cx 43. All transfected cell lines (3 clones transfected with the cx 26 gene, 2 clones with cx 40, and 1 with cx 43) showed establishment of gap junctional intercellular communication (GJIC). Two of the cx 26-transfected clones showed significantly slower growth compared with the parental HeLa cells. When transfectants were grown in soft agar, the three cx 26-transfected clones grew much less than the other transfectants and parent HeLa cells. When injected into nude mice, the two cx 26 clones which exhibited the highest amount of cx 26 transcript induced almost no tumors, whereas other transfectants, including the cx 26 clone which exhibited the lowest amount of cx 26 transcript, were tumorigenic. Among transfectants of various connexin genes, there was no good inverse correlation between their GJIC and tumorigenicity. GJIC levels were significantly higher in tumors induced in nude mice by clone cx 26 A and E transfectants. These results suggest that all of the connexin genes examined could induce recovery of GJIC of HeLa cells, but only the cx 26 gene exerts strong negative growth control on HeLa cells; thus, this connexin gene may have different functions from other connexin genes.

Differential effect of subcellular localization of communication impairing gap junction protein connexin43 on tumor cell growth in vivo.

There is a large body of evidence suggesting the connexin gap junction proteins appear to act as tumor suppressors, and their tumor inhibitory effect is usually attributed to their main function of cell coupling through gap junctions. However, some cancer cells (e.g. the rat bladder carcinoma BC31 cell line) are cell-cell communication proficient. Using specific site-directed mutagenesis in the third membrane-spanning (3M) domain of connexin43 (Cx43), we abolished the intrinsic gap junction intercellular communication (GJIC) in BC31 cells either by closing the gap junctional channels or by disruption of the transport of connexin complexes to the lateral membrane. Clones of BC31 cells transfected with a dominant negative Cx43 mutant giving rise to gap junctional channels, permeable only for a small tracer (neurobiotin), displayed accelerated growth rate in vivo, showing the critical role of selective gap junctional permeability in the regulation of cell growth in vivo. The use of other dominant-negative mutants of Cx43 also suggested that the effect of impaired communication on the tumorigenicity of cancer cells depends on the subcellular location of connexin. Inhibition of intrinsic GJIC in BC31 cells by sequestering of Cx protein inside the cytoplasm, due to expression of dominant-negative transport-deficient Cx43 mutants, did not significantly enhance the growth of transfectants in nude mice, but occasionally slightly retarded it. In contrast, augmentation of GJIC in BC31 cells by forced expression of wild-type Cx43, or a communication-silent mutant, fully suppressed tumorigenicity of these cells. Overall, these results show that cell coupling is a strong, but not the sole, mechanism by which Cx suppresses growth of tumorigenic cells in vivo; a GJIC-independent activity of Cx proteins should be considered as another strong tumor-suppressive factor.

Gap junctional intercellular communication and cell proliferation during rat liver carcinogenesis.

During multistage liver carcinogenesis, there is a sequential decrease in gap junctional intercellular communication (GJIC), associated with reduced expression of a major liver gap-junction protein (connexin 32). There are also several lines of evidence indicating that the induction of cell proliferation plays an important role during liver carcinogenesis. The relationship between GJIC and cell proliferation and their roles in liver carcinogenesis are not yet known. Results from various experiments suggest that there is a close relationship between the inhibition of GJIC and stimulation of liver cell proliferation. However, our results also suggest that different stimuli may affect cell proliferation and GJIC differentially by different mechanisms.

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.

The role of gap junctional intercellular communication (GJIC) disorders in experimental and human carcinogenesis.

There is a growing body of evidence supporting the etiologic implication of gap junctional intercellular communication disorders in carcinogenesis. Substantial progress has recently been made both in molecular biology of gap junction and in the field of cancer research. They provide new insights and conceptions of gap junctional disorders in tumor pathology. Modern understanding of the structure, function and regulation of gap junctions, as well as putative mechanisms of its disorders in human and experimental carcinogenesis are discussed in this review with particular emphasis on fast-moving aspects of this problem.

Role of blocked gap junctional intercellular communication in non-genotoxic carcinogenesis.

Gap junctional intercellular communication mediates the transfer of small molecules from the cytoplasm of one cell to that of neighbouring cells. Connexins are the proteins that form the channels responsible for this type of communication. Aberrant expression and function of connexins are often found in cells exposed to tumor-promoting agents and during carcinogenesis, both in cell culture systems and in tissues freshly removed directly from patients and exposed animals. Transfection of connexin genes into tumorigenic cells often exerts negative growth control, suggesting that connexins act as a family of tumor-suppressor genes. Connexin gene mutations appear to be the cause of two human diseases, i.e. X-linked Charcot-Marie-Tooth syndrome and visceroatrial heterotaxia. Connexin genes are therefore important for the maintenance of homeostasis and thus their dysfunction could lead to various forms of disease.

Connexin gene mutations in human genetic diseases.

Rapid advances in understanding the molecular biology of the gap junctional proteins - connexins (Cx) - have revealed that these proteins are indispensable for various cellular functions. Recent findings that mutational alterations of Cx genes leads to several quite different human diseases provide additional evidence that these proteins possess several not yet fully understood functions. Many different mutations of Cx32 have been found in the hereditary peripheral neuropathy - X-linked Charcot-Marie-Tooth syndrome and several mutations of Cx26 and Cx31 have been detected in deafness. Individual mutations of Cx46, Cx50 and Cx43 have been found in cataract or heart malformations. In this review, we analyzed the functional importance of mutations of different Cx described in different human diseases. Topological comparison of mutations in different Cx species has revealed several hot spots, where mutations are common for two different Cx or diseases. The value of Cx mutations associated with diseases for understanding Cx functions is discussed.

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.

[A method for inducing gallbladder cancer in Syrian hamsters]. / Metodika indutsirovaniia raka zhelchnogo puzyria u siriiskikh khomiakov.

The 8-10 weeks male Syrian hamsters were inserted beeswax pellets into the gallbladder, the pellets contained 5 mg of 3-methylcholanthrene (group IV). In control groups the animals were inserted intravesical beeswax pellets or subjected to a sham-operation (group I, II, III). The experiment lasted for 34 weeks, the first tumours were obtained 21 weeks after the operation. The tumours of gallbladder were obtained in group IV in 23 of 42 animals which lived for more than a month (54.2%). The first results of the experiment allow estimating positively the method, which permits due to the exact performance of the described details obtaining a number of tumours sufficient for studying the tumours of the gallbladder in a comparatively short period.

[The scientific and clinical value of molecular genetic changes in the intercellular gap junctions observed in colon cancer]. / Znachenie molekuliarno-geneticheskikh izmenenii mezhkletochnykh shchelevykh kontaktov pri rake tolstoi kishki.

Colon cancer is one of the most widespread pathologies with high mortality due to recurrence and metastasis. The molecular methods of diagnosis, prognostication and biotherapy in colorectal cancer enjoyed a rapid progress during the recent decades. The hypothesis on the key role of impaired intercellular gap junctions in the onset and progression of malignant tumors is a promising trend in carcinogenesis research. We have recently discovered a variety of tumor-specific mutations of connexin 43 gene in advanced colorectal cancer (Oncogene. -2002.-Vol.21, No.32-pp.4992-4996), which confirms the above hypothesis in malignant tumor progression. We believe that further studies of connexins' mutation changes in tumor growth is a promising trend in research of its etiology and pathogenesis and in designing new methods of diagnostics and treatment of colonic and other gastrointestinal cancers.

[Morphogenesis of experimental cancer of the large intestine (according to the results of scanning electron and light microscopy)]. / Morfogenez éksperimental'nogo raka tolstoi kishki (po dannym rastrovoi élektronnoi i svetovoi mikroskopii).

Colon tumors induced by 1.2-dimethylhydrazine in mice were studied by scanning electron microscopy followed by light microscopy. The following morphological types of tumors were demonstrated: adenomatous polyps (82%) with folded and smooth patterns of the surface structure, hyperplastic polyps (13.8%), endophytic carcinoma (3.7%), and few villous tumors. In the majority of the cases carcinoma developed due to malignisation of morphologically benign adenomatous and more rarely hyperplastic polyps. Endophytic carcinoma developed without benign precursors. Villous tumors had a high potential for malignisation. All these kinds of tumors arose in diffuse hyperplastic colon mucosa.

[Sensitivity of different lines of mice to the carcinogenic action of 1,2-dimethylhydrazine]. / O chuvstvitel'nosti myshei razlichnykh linii k kantserogennomu deistviiu 1,2-dimetilgidrazina.

Female mice of lines TBA, C57BL/6, BALB/c, and hydrids F1 (CBAxC57BL/6) were given 1,2-dimethylhydrazine (DMH) in a dose of 16 mg/kg 20 times weekly. Mice of line BALB/c proved to be mostly sensitive to a carcinogenic effect of DMH (the most low survival rate, the appearance of tumors in the anal region 5 weeks earlier, and much greater probability of their occurrence compared with other lines, extensive tumor invasion of the large intestine). The most low sensitivity to a carcinogenic action of DMH was noted in mice of line C57BL/6. It was found possible to induce uterine sarcomas by DMH in hybrids F1 (CBAxC57BI/6) in a comparatively high per cent of cases (15.4%). Morphological changes in the liver were noted in DMH exposed mice of all lines (dystrophy, cystic cavities, the outgrowth of bile ducts and the connective tissue), which were mostly pronounced in mice of line C57BL/6 (in one case it was adenocarcinoma). The genetic factor is suggested to be of certain importance in the sensitivity to DMH carcinogenic effect.

[The character of expression of keratins 8 and 19 in normal and neoplastic enterocytes of the large intestine]. / Kharakter ékspressii keratinov 8 i 19 v normalnykh i opukholevykh énterotsitakh tolstoi kishki.

Synthesis of individual keratins (Nos. 8 and 19) was shown to continue in the simple epithelium of tumor cells by application of monoclonal antibodies in 20 cases of different patterns of human colonic malignancies. No correlation between the synthesis and degree of cataplasia of said cells was found. The increase in manifestations of cellular and structural anaplasia in tumor was matched by the enhanced intensity of cells' staining with antibodies. Application of said procedure in oncomorphological practice helps reliably evaluate the real extent of tumor spreading and detects invasion which other microscopic methods fail to do.

[The induction of cancer of the large intestine in Macaca fascicularis monkeys with 1,2-dimethylhydrazine]. / Induktsiia raka tolstoi kishki u obez'ian Macaca fascicularis 1,2-dimetilgidrazinom.

Continuous subcutaneous administration of 16 mg/kg body weight 1,2-dimethylhydrazine three times a month (total dose--1080-3696 mg) to Macaca fascicularis monkeys induced cancer invariably confined to the colon within 34-47 weeks. Biologic, clinical, histologic features and natural course of the tumor proved similar to those of its human counterpart.

[New insights in oncology: epigenetics and cancer stem cells]. / Nouvelles perspectives en oncologie : épigénétique et cellules souches cancéreuses.

Cancer is a multi-etiologic, multistage disease with a prevalent genetic component, which happens when a large number of genes, critical for cell growth, death, differentiation, migration, and metabolic plasticity are altered irreversibly, so as to either "gain" (oncogenes) or "lose" (tumour suppressors) their function. Recent discoveries have revealed the previously underestimated etiologic importance of multiple epigenetic, that is to say, reversible factors (histone modifications, DNA methylation, non-coding RNA) involved in the transcriptional and post-transcriptional regulation of proteins, indispensable for the control of cancerous phenotype. Stable alterations of epigenetic machinery ("epimutations") turn out to play a critical role at different steps of carcinogenesis. In addition, due to substantial recent progress in stem cell biology, the new concept of cancer stem cells has emerged. This, along with newly discovered epigenetic cancer mechanisms, gives rise to a hope to overcome radio- and chemo-resistance and to eradicate otherwise incurable neoplasms.

Assessment of transformed properties in vitro and of tumorigenicity in vivo in primary keratinocytes cultured for epidermal sheet transplantation.

Epidermal keratinocytes are used as a cell source for autologous and allogenic cell transplant therapy for skin burns. The question addressed here is to determine whether the culture process may induce cellular, molecular, or genetic alterations that might increase the risk of cellular transformation. Keratinocytes from four different human donors were investigated for molecular and cellular parameters indicative of transformation status, including (i) karyotype, (ii) telomere length, (iii) proliferation rate, (iv) epithelial-mesenchymal transition, (v) anchorage-independent growth potential, and (vi) tumorigenicity in nude mice. Results show that, despite increased cell survival in one keratinocyte strain, none of the cultures displayed characteristics of cell transformations, implying that the culture protocol does not generate artefacts leading to the selection of transformed cells. We conclude that the current protocol does not result in an increased risk of tumorigenicity of transplanted cells.

[Immunomorphological study of 1,2-dimethylhydrazine-induced carcinomas of the large intestine in rats using monoclonal antibodies to intermediate filament proteins]. / Immunomorfologicheskoe izuchenie kartsinom tolstoi kishki krys, indutsirovannykh 1,2-dimetilgidrazinom, s ispol'zovaniem monoklonal'nykh antitel k belkam promezhutochnykh filamentov.

Cryostatic sections of rat large bowel tumors induced by 1,2-dimethylhydrazine were stained with monoclonal antibodies against different proteins of intermediate filaments: (a) against prekeratin (mol. mass 49 000, PK49) found in many epithelial cells and (b) against vimentin, a constituent of intermediate filaments of mesenchymal cells. Immunofluorescence study showed that large bowel tumor cells as well as normal cells of this organ contain PK49 but not vimentin. High sensitivity of the method allowed one to clearly identify small invasive nodules and groups of tumor cells not visible in usual histologic preparations. Moreover, in some cases single atypical tumor cells were identified in tumor stroma and in the submucosal layer underlying the tumor, that were indistinguishable from normal mesenchymal cells at the light microscopy level.

Distribution of laminin and collagen type IV in rat colon tumors induced by 1,2-dimethylhydrazine.

Immunofluorescent distribution of basement membrane components laminin and collagen type IV was studied in 51 rat colon tumors induced by 1, 2-dimethylhydrazine. In normal colonic mucosa, adenomas and carcinomas in situ continuous basement membranes were present, while in adenocarcinomas they were altered to different extents. An uncoordinated loss, or dissociation, of the two markers studied was found: the degree of collagen type IV loss was often much higher than that of laminin in the same tumor. These data suggest that a reliable determination of cancer invasion by monitoring basement membrane alteration requires the use of several basement membrane markers.