Appearance of differentiation characteristics (induction of Ah-receptor-dependent genes) during cultivation of transformed cell clone K8 from embryonic rat fibroblasts.

The differentiation status of fibroblasts can be characterized by their ability to induce Ah-receptor-dependent genes. The ability to induce Ah-receptor-dependent genes encoding cytochrome P450 isoforms, Ah-receptor repressor, and NADPH-quinine oxidoreductase were studied in the transformed cell clone K8 obtained from immortalized embryonic rat fibroblasts by treatment with benzo(a)pyrene and in the parental clone F27. Treatment with benz(a)anthracene did not induce the genes in the transformed clone K8 on passages 4-14, but the induction was recorded in the transformed clone beginning from the 16th passage and later, whereas in F27 cells the induction was observed throughout the experiment. Induction levels of mRNA of the induction-regulating genes encoding the Ah-receptor and Ah receptor nuclear translocator were similar in F27 cells and in the transformed cell clone K8 in both early and late passages. Electrophoretic mobility shift assay showed that in clone K8 transmission of the induction signal was disturbed in the early passages before interaction of the activated Ah-receptor with the recognizing region of DNA. Possible mechanisms responsible for the absence of induction in the early passages in the transformed cells are discussed.

9-Aminoacridine-based anticancer drugs target the PI3K/AKT/mTOR, NF-kappaB and p53 pathways.

Acquisition of a transformed phenotype involves deregulation of several signal transduction pathways contributing to unconstrained cell growth. Understanding the interplay of different cancer-related signaling pathways is important for development of efficacious multitargeted anticancer drugs. The small molecule 9-aminoacridine (9AA) and its derivative, the antimalaria drug quinacrine, have selective toxicity for tumor cells and can simultaneously suppress nuclear factor-kappaB (NF-kappaB) and activate p53 signaling. To investigate the mechanism underlying these drug activities, we used a combination of two-dimensional protein separation by gel electrophoresis and mass spectrometry to identify proteins whose expression is altered in tumor cells by 9AA treatment. We found that 9AA treatment results in selective downregulation of a specific catalytic subunit of the phosphoinositide 3-kinase (PI3K) family, p110 gamma. Further exploration of this observation demonstrated that the mechanism of action of 9AA involves inhibition of the prosurvival AKT/mammalian target of rapamycin (mTOR) pathway that lies downstream of PI3K. p110 gamma translation appears to be regulated by mTOR and feeds back to further modulate mTOR and AKT, thereby impacting the p53 and NF-kappaB pathways as well. These results reveal functional interplay among the PI3K/AKT/mTOR, p53 and NF-kappaB pathways that are frequently deregulated in cancer and suggest that their simultaneous targeting by a single small molecule such as 9AA could result in efficacious and selective killing of transformed cells.

Regulation of matrix metalloproteinase-9 transcription in squamous cell carcinoma of uterine cervix: the role of human papillomavirus gene E2 expression and activation of transcription factor NF-kappaB.

Matrix metalloproteinase-9 (MMP-9) plays an important role in initiation and progression of squamous cell carcinoma (SCC) of human uterine cervix. Regulation of MMP-9 expression in such tumors is insufficiently studied. Involvement of the human papillomavirus (HPV) gene E2 and transcription factor NF-kappaB in the regulation of MMP-9 transcription has been shown in some model systems and types of malignant tumors. The present work was mainly designed to reveal a possible role of the HPV gene E2 and transcription factor NF-kappaB in the induction of MMP-9 expression in SCC. Specimens of tumor and corresponding adjacent normal tissue from 26 patients with SCC of the uterine cervix were studied. The intact E2 frame was observed in 19 of 26 (73.1%), the E2 gene mRNA was expressed in 10 of 15 (66.7%), NF-kappaB was activated in 17 of 23 (73.9%), and the expression of MMP-9 mRNA was recorded in 10 of 20 (50%) of the informative cases. The MMP-9 transcription did not correlate with gene E2 status, but in all cases correlated with the activation of NF-kappaB transcription factor (10 of 10 vs. 5 of 10 MMP-9-negative cases, p = 0.016). Thus, the NF-kappaB role has been proved in the regulation of MMP-9 transcription in SCC. There was no correlation of the E2 status and MMP-9 expression with clinical/morphological characteristics of the tumors: size, local invasiveness, metastasizing into regional lymph nodes, and level of differentiation. The high intensity of NF-kappaB activation correlated with low degree of differentiation of the tumors studied (p = 0.044). These findings suggested that NF-kappaB should be a molecular factor of the poor prognosis of human SCC.

Benzo[a]pyrene-dependent activation of transcription factors NF-kappaB and AP-1 related to tumor promotion in hepatoma cell cultures.

The activation by the carcinogenic polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BP) of transcription factors NF-kappaB and AP-1 in hepatoma 27 and HepG2 cell cultures was studied. In contrast to the hepatoma HepG2 cells, cytochrome P450 isoforms and Ah-receptor are not expressed in the hepatoma 27 cells. The transcription factor NF-kappaB was activated only in the hepatoma 27 cells by BP treatment but not by its noncarcinogenic isomer benzo[e]pyrene (BeP). Conversely to NF-kappaB activation the transcription factor AP-1 was activated in the hepatoma HepG2 cells by cell treatment with BP but not in the hepatoma 27 cells. It is concluded that the NF-kappaB activation is caused by nonmetabolized BP molecule and not related to activation of the Ah-receptor. The transcription factor AP-1 seems to be activated as a result of the interaction of BP with the Ah-receptor. The realization of tumor promotion stage by carcinogenic PAHs treatment in dependence on the cytochrome P450 and Ah-receptor levels in the initiated cells is discussed.

Allelic imbalance and instability of microsatellite loci on chromosome 1p in human non-small-cell lung cancer.

The mapping of allelic loss on the short arm of chromosome 1 has been performed in non-small-cell lung cancer. We used a set of 11 microsatellite loci spanning 1p to examine the frequency of allelic imbalance in a panel of 58 tumours. Fifty-one of 58 (87.9%) cases have shown somatic allelic loss at one or more loci tested. The two shortest regions of the overlap (SRO) of the deletions have been identified: SRO 1 at 1p13.1 and SRO 2 at 1p32-pter. Allelic losses at these regions have been compared among adenocarcinoma and squamous cell carcinoma and no difference has been found. In contrast to SRO 1, deletions at SRO 2 significantly correlated with advanced stage of the disease as well as post-operative metastasizing and relapse. These data may suggest that SRO 1 and SRO 2 can harbour tumour-supressor genes (TSGs) involved in different stages of NSCLC development. SRO 2 is still quite large and its refined mapping should help attempts to clone and identify the putative TSG(s). Microsatellite instability (replication errors) affecting only 6 (10.3%) of 58 tumour samples is an infrequent genetic alteration at the loci tested.

[Comparative characteristics of cyclodextrin glycosyltransferases from various groups of microorganisms]. / Sravneitel'naia kharakteristika tsiklodekstringlikoziltransferaz razlichnykh grupp mikroorganizmov.

Cyclodextrin glycosyltransferases (CGT-ase, 1.4-alpha-glucanotransferase, cyclizing, EC 2.4.1.19) produced by some thermophilic, alkalophilic and mesophilic bacterial strains, were isolated and characterized. It was shown that thermophilic and mesophilic CGT-ases represent a mixture of alpha-, beta- and gamma-cyclodextrins (CD), alpha-cyclodextrin being the predominant component. Alkalophilic enzymes produce only beta-CD and are able to produce CD not only from starch but also from maltose, melibiose, maltotriose, amylose and amylopectin. The optimal conditions for the catalytic activity of the enzymes were determined. It was found that calcium, magnesium and zinc ions have a beneficial effect on the specific activity of these enzymes. The amino acid composition of the enzymes was studied.