An optimized protocol for electroporation is described which is suitable for introducing antibodies into mammalian cells. The method results in the uptake of detectable amounts of antibodies in 80% of the cells and in 40% large amounts are introduced. As an example, cell cycle activity (transition from the G1 to S phase) was inhibited by the introduction of monoclonal antibodies against G1-specific cyclin D1 into CV-1 and MCF7 cells. This specific antibody mediated inhibition of cellular function did not affect the viability of the cells since they recovered from the inhibition after some time. While approaching the efficiency of microinjection, the new protocol for electroporation of antibodies additionally permits treatment of the larger number of cells which are required for biochemical analyses.

• MeSH
• buněčné linie MeSH
• cyklin D1 MeSH
• cykliny imunologie   MeSH
• elektroporace * MeSH
• fluorescenční protilátková technika MeSH
• Haplorrhini MeSH
• imunoglobulin G imunologie   MeSH
• kompetitivní vazba MeSH
• lidé MeSH
• mikroinjekce MeSH
• monoklonální protilátky imunologie   metabolismus   MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• nádory prsu MeSH
• onkogenní proteiny imunologie   MeSH
• S fáze imunologie   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

A cascade of events is triggered upon the addition of growth factor to quiescent mammalian cells, which ultimately restarts proliferation by inducing the transition from G0/G1 to S-phase. We have studied cyclin D1, a putative G1 cyclin, in normal diploid human fibroblasts. Cyclin D1 accumulated and reached a maximum level before S-phase upon the addition of serum to quiescent cells. The protein was localized to the nucleus, and it disappeared from the nucleus as cells proceeded into S-phase. Microinjection of anti-cyclin D1 antibodies or antisense plasmid prevented cells from entering S-phase, and the kinetics of inhibition showed that cyclin D1 is required at a point in the cell cycle earlier than cyclin A. These results demonstrate that cyclin D1 is a critical target of proliferative signals in G1.

• MeSH
• antisense elementy (genetika) MeSH
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• časové faktory MeSH
• cyklin D1 MeSH
• cykliny fyziologie   metabolismus   MeSH
• DNA biosyntéza   MeSH
• fibroblasty cytologie   MeSH
• fluorescenční protilátková technika MeSH
• G1 fáze * fyziologie   MeSH
• lidé MeSH
• mikroinjekce MeSH
• onkogenní proteiny fyziologie   metabolismus   MeSH
• plazmidy MeSH
• plíce cytologie   fyziologie   ultrastruktura   MeSH
• S fáze * fyziologie   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• histologické techniky MeSH
• imunohistochemie MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• myši MeSH
• nádorový supresorový protein p53 analýza   imunologie   MeSH
• nádory chemie   MeSH
• rekombinantní proteiny imunologie   MeSH
• specificita protilátek MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

• MeSH
• dospělí MeSH
• ELISA MeSH
• karcinom in situ chemie   MeSH
• lidé MeSH
• metastázy nádorů MeSH
• nádorový supresorový protein p53 analýza   MeSH
• nádory prsu chemie   MeSH
• nádory genetika   chemie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH

• MeSH
• buněčný cyklus genetika   MeSH
• imunoblotting MeSH
• lidé MeSH
• myši MeSH
• nádorové proteiny genetika   MeSH
• nádory enzymologie   genetika   patologie   MeSH
• proteinkinasa CDC2 genetika   MeSH
• proteinkinasy genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

Overexpression of c-erbB-2 occurs in 60% of in situ and 25% of infiltrating ductal carcinomas. We have previously found very strong associations between immunohistochemical staining for c-erbB-2 and histological pattern and nuclear size in ductal carcinoma in situ (DCIS) and less strong correlation with proliferative activity. In a further study of infiltrating ductal carcinomas we have found that, in addition to tumours arising from c-erbB-2 positive, large celled, rapidly proliferating, comedo carcinomas and c-erbB-2 negative small celled cribriform/micropapillary carcinomas with a low proliferative rate, there is a third group of c-erbB-2 negative tumours with large nuclei and variable proliferative activity. These latter tumours are not seen in pure DCIS suggesting that they have a very transient in situ stage. Therefore, although in pure DCIS c-erbB-2 positively appears to be associated with tumours with a greater invasive potential, and c-erbB-2 negativity with tumours having a more favourable prognosis, the latter is not necessarily true in infiltrating disease.

• MeSH
• buněčné jádro patologie   MeSH
• intraduktální neinfiltrující karcinom * chemie   patologie   MeSH
• invazivní růst nádoru MeSH
• karcinom in situ * chemie   patologie   MeSH
• lidé MeSH
• nádory prsu * chemie   patologie   MeSH
• protoonkogenní proteiny * analýza   MeSH
• receptor erbB-2 MeSH
• S fáze fyziologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH

• MeSH
• antigeny transformující polyomavirové MeSH
• geny p53 genetika   MeSH
• geny retinoblastomu genetika   MeSH
• imunokomplex MeSH
• lidé MeSH
• mutace MeSH
• onkogenní proteiny virové MeSH
• opičí virus SV40 imunologie   MeSH
• Check Tag
• lidé MeSH

When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the phenotype of the common luminal epithelial cell found in the terminal ductal lobular units. Luminal epithelial cells cultured from milk, which have limited proliferative potential, have now been immortalized by introducing the gene encoding simian virus 40 large tumor (T) antigen. Infection with a recombinant retrovirus proved to be 50-100 times more efficient than calcium phosphate transfection, and of the 17 cell lines isolated, only 5 passed through a crisis period as characterized by cessation of growth. When characterized by immunohistochemical staining with monoclonal antibodies, 14 lines showed features of luminal epithelial cells and of these, 7 resembled the common luminal epithelial cell type in the profile of keratins expressed. These cells express keratins 7, 8, 18, and 19 homogeneously and do not express keratin 14 or vimentin; a polymorphic epithelial mucin produced in vivo by luminal cells is expressed heterogeneously and the pattern of fibronectin staining is punctate. Although the cell lines have a reduced requirement for added growth factors, they do not grow in agar or produce tumors in the nude mouse. When the v-Ha-ras oncogene was introduced into two of the cell lines by using a recombinant retrovirus, most of the selected clones senesced, but one entered crisis and emerged after 3 months as a tumorigenic cell line.

• MeSH
• antigeny transformující polyomavirové * genetika   MeSH
• buněčné dělení MeSH
• buněčné linie MeSH
• DNA virů genetika   MeSH
• epitelové buňky MeSH
• fluorescenční protilátková technika MeSH
• genetické vektory MeSH
• keratiny metabolismus   MeSH
• lidé MeSH
• mateřské mléko * cytologie   MeSH
• myši nahé MeSH
• myši MeSH
• onkogenní protein p21(ras) genetika   MeSH
• opičí virus SV40 genetika   MeSH
• prsy * cytologie   MeSH
• Retroviridae genetika   MeSH
• Southernův blotting MeSH
• techniky in vitro MeSH
• virová transformace buněk * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

A series of 14 new mouse monoclonal antibodies (MAbs) to keratins is described and the data suggesting their potential value in the differential diagnosis of human tumours are reported. The specificities of individual MAbs of the 'C-series' presented here range from monospecificity for keratin No. 7 (MAbs C-18, C-35, C-62, and C-68), keratin No. 8 (MAbs C-15, C-43, and C-15), and keratin No. 18 (MAbs C-04 and C-08) up to the broadly reacting 'pan-keratin' MAb C-11, with the target epitopes of the remaining four MAbs being shared by different pairs of keratin polypeptides. The results of the biochemical characterization of the MAbs, together with their immunohistochemical staining patterns on frozen as well as on paraffin sections of normal human tissues, suggest that they represent a significant contribution to the growing list of anti-keratin MAbs applicable in both research and routine diagnostic pathology. The immunohistochemical examination of a wide range of human neoplasms with the new MAbs not only confirmed their value in making distinctions between carcinomas, on the one hand, and lymphomas, and gliomas, on the other, but also verified the possibility of more subtle subdivisions within the group of adenocarcinomas and their metastases. Furthermore, the identification of small subsets of breast carcinomas with decreased levels or apparent loss of the keratin No. 7 polypeptide and some cases of stomach carcinoma with apparently induced expression of this keratin suggests that such 'exceptions' must be considered when using keratin spectra as one of the criteria in differential diagnosis.

• MeSH
• 2D gelová elektroforéza MeSH
• buněčné linie MeSH
• epitel imunologie   MeSH
• fluorescenční protilátková technika MeSH
• imunoblotting MeSH
• keratiny analýza   imunologie   MeSH
• lidé MeSH
• monoklonální protilátky diagnostické užití   imunologie   MeSH
• myši MeSH
• nádory chemie   imunologie   patologie   MeSH
• specificita protilátek MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

• MeSH
• adenovirové proteiny E1A * fyziologie   MeSH
• antigeny transformující polyomavirové * fyziologie   MeSH
• buněčné linie MeSH
• buněčný cyklus * MeSH
• cyklin D1 MeSH
• cykliny fyziologie   metabolismus   MeSH
• lidé MeSH
• lidské adenoviry genetika   MeSH
• onkogenní proteiny virové * fyziologie   MeSH
• onkogenní proteiny * fyziologie   MeSH
• onkogeny * MeSH
• opičí virus SV40 genetika   MeSH
• Papillomaviridae genetika   MeSH
• regulace exprese virových genů MeSH
• retinoblastomový protein * fyziologie   MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH