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Presence of dysfunctional senescent hepatocytes is a hallmark feature of liver cirrhosis which finally culminates in liver cancer. We now report the presence of senescent hepatocytes (p21 and p53 positive) in the vicinity of infiltrated immune cells in hepatocellular carcinoma tissue specimens by immunohistochemistry. Hence, we evaluated in vitro, the relevance of senescent hepatoma cells in altering the fate of monocytes and neutrophils by assaying for macrophage polarization and extracellular trap (NETs) formation, respectively. Premature senescence was induced in hepatoma cells (HepG2 and Huh7 cells) by treating cells with doxorubicin. Senescent hepatoma cells showed strong inflammatory phenotype with induced expression of cytokines (IL1β, IL6, IL8 and IL13) as evaluated by flow cytometry. The senescent secretome from hepatoma cells when incubated with healthy monocytes caused it to differentiate predominantly towards M2 fate (CD80

Background Decompensated cirrhosis patients are more prone to bacterial infections. Myeloid derived suppressor cells (MDSCs) expand in sepsis patients and disrupt immune cell functions. GM-CSF therapy helps in restoring immune cell functions and resolve infections. Its role in MDSCs modulation in cirrhotic with sepsis is not well understood. Methods 164 decompensated cirrhotic; 62 without(w/o), 72 with sepsis and 30 with sepsis treated with GM-CSF and 15 healthy were studied. High-dimensional flow cytometry was performed to analyse MDSCs, monocytes, neutrophils, CD4 T-cells and Tregs at admission, day3 and 7. Ex-vivo co-cultured MDSCs with T-cells were assessed for proliferation and apoptosis of T-cells, differentiation to T-regs. Plasma factors and mRNA levels were analysed by cytokine-bead assay and qRT-PCR. Results Frequency of MDSCs and T-regs were significantly increased (p=0.011, and p=0.02) with decreased CD4 T-cells(p=0.01) in sepsis than without sepsis and HC (p=0.000, p=0.07 and p=0.01) at day0, and day7. In sepsis patients, MDSCs had increased IL-10, Arg1 and iNOS mRNA levels (p=0.016, p=0.049 and p=0.06). Ex-vivo co-cultured MDSCs with T-cells drove T-cell apoptosis (p=0.03, p=0.03) with decreased T-cell proliferation and enhanced FOXP3+ expression (p=0.05 and p=0.05) in sepsis compared to no sepsis at day0. Moreover, blocking the MDSCs with inhibitors suppressed FOXP3 expression. GM-CSF treatment in sepsis patients significantly decreased MDSCs and FOXP3+Tregs but increased CD4 T-cell functionality and improved survival. Conclusion MDSCs have immunosuppressive function by expanding FOXP3+ Tregs and inhibiting CD4+ T-cell proliferation in sepsis. GM-CSF treatment suppressed MDSCs, improved T-cell functionality and reduced Tregs in circulation.

Background Targeting cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) is difficult because of their similarities with normal stem cells (NSCs). EpCAM can identify CSCs from EpCAM+AFP+HCC cases, but is also expressed on NSCs. We aimed to distinguish the two using integrated protein, mRNA and miRNA profiling. Methods iTRAQ based protein profiling and Next Generation Sequencing (NGS) was performed on EpCAM+/EpCAM- cells isolated from HCC (Ep+CSC, Ep- HCC) and EpCAM+ cells from non-cancerous/non-cirrhotic control liver tissues (Ep+NSC). Validations were done using qRT-PCR, flowcytometry and western blotting followed by in vitro and in vivo functional studies. Results 11 proteins were overexpressed (>3 fold) in Ep+CSCs compared to Ep- HCC and Ep+NSC cells. However, RNA-sequencing confirmed the Ep+CSC specific up-regulation of only HSPA8, HNRNPC, MPST and GAPDH mRNAs among these. Database search combined with miRNA profiling revealed Ep+ CSC specific down-regulation of 29 miRNAs targeting these four genes. Of these, only miR-26b-5p was found to target both HSPA8 and EpCAM. Validation of HSPA8 overexpression and miR-26b-5p down-regulation followed by linear regression analysis established a negative correlation between the two. Functional studies demonstrated that reduced miR-26b-5p expression increased the spheroid formation, migration, invasion and tumourigenicity of Ep+ CSCs. Furthermore, anti-miR-26b-5p increased the number of Ep+ CSCs with a concomitant overexpression of stemness genes and reduction of proapoptotic protein BBC3, which is a known substrate of HSPA8. Conclusion miR-26b-5p imparts metastatic properties and helps in maintenance of Ep+ CSCs via HSPA8. Thus, miR-26b-5p and HSPA8 could serve as molecular targets for selectively eliminating the Ep+ CSC population in human HCCs.

Background: Myeloid-derived suppressor cells (MDSCs) are immunosuppressive in nature, originate in the bone marrow, and are mainly found in the blood, spleen, and liver. In fact, liver acts as an important organ for induction and accumulation of MDSCs, especially during infection, inflammation, and cancer. In humans and rodents, models of liver diseases revealed that MDSCs promote regeneration and drive the inflammatory processes, leading to hepatitis, fibrogenesis, and cirrhosis, ultimately resulting in hepatocellular carcinoma. Summary: This brief review is focused on the in-depth understanding of the key molecules involved in the expansion and regulation of MDSCs and their underlying immunosuppressive mechanisms in liver diseases. Key Message: Modulated MDSCs can be used for therapeutic purposes in inflammation, cancer, and sepsis.

SIRT7 belongs to the family of “NAD+ dependent deacetylases” called Sirtuins. In the present work we report a novel role of SIRT7 in regulating cellular polarity. SIRT7 overexpression in immortalized mouse fibroblasts (NIH3T3) induced epithelial transition. This transition was accompanied by typical N- to E- cadherin transition, stabilization of β-catenin, and the downregulation of transcription factors responsible for maintenance of mesenchymal phenotype (Snail, Slug, and Zeb1). Interestingly, a subpopulation of cells overexpressing SIRT7 exhibited an intermediate stage between mesenchymal and epithelial characters. Transformed epithelial cells showed a loss of heterochromatisation as evidenced by a loss of HP1α and H3K9 dimethylation staining. In conclusion, we report a role of SIRT7 in mesenchymal cells, which may have implications for health and disease.

Sirtuins belong to the family of class III histone deacetylases; its role in neoplasia is controversial as both tumor-suppressive and promoting functions have been reported. There are very few reports available, where expressions of sirtuin isoforms are comprehensively analyzed during neoplasia. Therefore, in the present study, the expression of SIRT1, SIRT2, and SIRT7 during different stages of cervical cancer progression was analyzed. The normal cervical epithelium showed feeble expression of sirtuin isoforms, SIRT1, SIRT2, and SIRT7. A significant increase in SIRT1 expression was noted in the cytoplasm as well as in the nucleus of proliferative layers of cervical epithelium in squamous intraepithelial lesions (SIL); however, in the squamous cell carcinomas (SCC), a heterogeneous pattern of SIRT1 expression varying from low to high was noted. A progressive increase in the expression of both SIRT2 and SIRT7 was noted during cancer progression in the following order: normal

The calcineurin-NFAT signaling pathway regulates cell proliferation, differentiation, and development in diverse cell types and organ systems. Deregulation of calcineurin-NFAT signaling has been reported in leukaemias and few solid tumors such as breast and colon. In the present study, we found elevated calcineurin protein levels and phosphatase activity in cervical cancer cell lines and depletion of the same attenuated cell proliferation. Additionally, nuclear levels of NFAT2, a downstream target of calcineurin, viz, was found elevated in human papillomavirus (HPV) infected cells, HeLa and SiHa, compared to the HPV negative cells, HaCaT and C33A, indicative of its higher DNA binding activity. The nuclear levels of both NFAT1 and NFAT3 remain unaltered implicating they have little role in cervical carcinogenesis. Similar to the in vitro studies, the HPV infected human squamous cell carcinoma specimens showed higher NFAT2 levels compared to the normal cervical epithelium. Depletion of NFAT2 by RNAi attenuated growth of SiHa cells. Overexpression of HPV16 oncoproteins viz, E6 and E7 increased NFAT2 expression levels and DNA binding activity, while knockdown of E6 by RNAi decreased the same. Briefly, we now report an activation of calcineurin-NFAT2 axis in cervical cancer and a novel role of HPV oncoprotein in facilitating NFAT2 dependent cell proliferation.

Sirtuins (SIRT) belonging to the NAD+ dependent histone deacetylase III class of enzymes have emerged as master regulators of metabolism and longevity. However, their role in prevention of organismal aging and cellular senescence still remains controversial. In the present study, we now report upregulation of SIRT2 as a specific feature associated with stress induced premature senescence but not with either quiescence or cell death. Additionally, increase in SIRT2 expression was noted in different types of senescent conditions such as replicative and oncogene induced senescence using multiple cell lines. Induction of SIRT2 expression during senescence was dependent on p53 status as depletion of p53 by shRNA prevented its accumulation. Chromatin immunoprecipitation revealed the presence of p53 binding sites on the SIRT2 promoter suggesting its regulation by p53, which was also corroborated by the SEAP reporter assay. Overexpression or knockdown of SIRT2 had no effect on stress induced premature senescence, thereby indicating that SIRT2 increase is not a cause of senescence; rather it is an effect linked to senescence-associated changes. Overall, our results suggest SIRT2 as a promising marker of cellular senescence at least in cells with wild type p53 status.

Epigenetic events play a prominent role during cancer development. This is evident from the fact that almost all cancer types show aberrant DNA methylation. These abnormal DNA methylation levels are not restricted to just a few genes but affect the whole genome. Previous studies have shown genome-wide DNA hypomethylation and gene-specific hypermethylation to be a hallmark of most cancers. Molecules like DNA methyltransferase act as effectors of epigenetic reprogramming. In the present study we have examined the possibility that the reprogramming genes themselves undergo epigenetic modifications reflecting their changed transcriptional status during cancer development. Comparison of DNA methylation status between the normal and cervical cancer samples was carried out at the promoters of a few reprogramming molecules. Our study revealed statistically significant DNA methylation differences within the promoter of DNMT3L. A regulator of de novo DNA methyltransferases DNMT3A and DNMT3B, DNMT3L promoter was found to have lost DNA methylation to varying levels in 14 out of 15 cancer cervix samples analysed. The present study highlights the importance of DNA methylation profile at DNMT3L promoter not only as a promising biomarker for cervical cancer, which is the second most common cancer among women worldwide, but also provides insight into the possible role of DNMT3L in cancer development.

Recent evidence suggests that K-ras protooncogene protein p21 may have a tumor-suppressive role in the context of development of lung adenocarcinoma. Levels of K-ras p21, raf-1, mitogen-activated protein kinases Erk 1 and 2, the phosphorylated-activated forms of Erk 1 and 2 (Erk 1P and 2P), and proliferating cell nuclear antigen (PCNA) were measured by immunoblotting in mouse lung tumors (5 to 9 mm in size) caused by N-nitrosodimethylamine (NDMA) and in control lungs. In tumors compared with normal lung, cell membrane-associated K-ras p21 was significantly decreased and cytosolic K-ras p21 increased. Total, membrane, and cytosolic raf-1 and Erk 1P and 2P were increased in tumors compared with normal lung. A single dose of 5 nmol/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) given after NDMA resulted in a significant 2.4-fold increase in tumor multiplicity. A significantly greater decrease in membrane-associated K-ras p21 and increase in total and membrane associated raf-1 occurred in the NDMA/TCDD tumors compared with the NDMA-only tumors. PCNA levels increased in tumors, a finding confirmed by immunohistochemistry, and correlated with tumor size after NDMA/TCDD treatment but not after NDMA only. The increase in raf-1 in the tumors was confirmed by immunohistochemistry, which also revealed an increase in raf-1-positive alveolar macrophages specifically associating with tumors from the earliest stages. These results suggest a possible tumor-suppressive function for K-ras p21 in lung and a positive role for raf-1 and Erk 1/2 in lung tumorigenesis. TCDD may promote tumors by contributing to downregulation of K-ras and stimulation of raf-1.

Growth factors bind to membrane receptor tyrosine kinases, resulting in autophosphorylation and subsequent binding to proteins with SH2 domains, including growth factor receptor-bound protein 2 (Grb2). Grb2 bridges receptors to tyrosine kinase substrates such as SHC and SOS, which in turn facilitate the activation of downstream signaling pathways, including Ras and mitogen-activated protein kinase (MAPK). Overexpression of Grb2 has been demonstrated in several types of neoplasia but has not been investigated in liver tumorigenesis. Here we investigated Grb2 expression in liver lesions in N-nitrosodimethylamine (NDMA)-treated Helicobacter hepaticus-infected and -noninfected A/J mice at 1 year of age. Previously, we reported (6) that infection promotes the development of these NDMA-initiated tumors. In controls, Grb2 immunostaining was absent from normal hepatic tissues, whereas the inflammatory lesions in infected livers were positive for cytoplasmic Grb2 in both hepatocytes and infiltrating leukocytes. All preneoplastic foci (7 of 7), 15 of 27 adenomas, and 3 of 7 carcinomas were positive for Grb2 by immunostaining in both infected and noninfected NDMA-initiated livers. Involvement of Grb2 was confirmed by immunoblotting of similarly infected mice at 9 to 18 months of age, showing a 2.5- to 3.3-fold increase in Grb2 protein in infected livers (p < 0.05 compared with uninfected controls) as well as in preneoplastic foci, adenomas, and carcinomas. These livers also showed a 2.5- to 2.8-fold increase in total Ras protein. The results suggest that upregulation of Grb2 is an early event in liver carcinogenesis, whether caused by the bacterial infection or by NDMA. Concomitant upregulation of Ras p21 would ensure transmission of amplified signal from growth factors via Grb2.

Codon 12 mutations are frequent in the Ki-ras oncogene in human lung adenocarcinomas, but the effects of these alterations have not been well characterized in lung epithelial cells. Murine primary lung tumors derived from peripheral epithelial cells also may present Ki-ras mutations and are useful models for study of early phases of tumor development. One hypothesis is that Ki-ras mutation and/or a Ki-ras p21 increase could enhance Ki-ras p21-GTP and cell-cycle stimulation through raf-1 and extracellularly regulated protein kinases (Erks). We examined lung tumors 1–7 mm in largest dimension initiated in male Swiss mice by N-nitrosodimethylamine for pathologic type, Ki-ras mutations and levels of total Ki-ras p21, Ki-ras p21 bound to GTP, raf-1, Erk1 and Erk2 and their phosphorylated (activated) forms, and proliferating cell nuclear antigen. Total Ki-ras p21 and activated ras-GTP were not significantly greater in tumors than in normal lung or in tumors with versus those without Ki-ras mutations. Carcinomas with Ki-ras mutations were significantly smaller than those without mutations. Carcinomas were significantly larger than adenomas only for tumors without mutations. High levels of Erk2 and correlation of Erk2 amount with ras-GTP were specific characteristics of tumors with Ki-ras mutations. Size of all tumors correlated with ras-GTP but not with proliferating cell nuclear antigen. Raf-1 was expressed mainly in alveolar macrophages in normal lung but was focally upregulated in papillary areas of some tumors. The results indicate that Ki-ras influences the characteristics of lung tumors, but a linear ras–raf–Erk–cell-cycle control sequence does not adequately characterize tumorigenic events in this model. Mol. Carcinog. 28:156–167, 2000. © 2000 Wiley-Liss, Inc.

The roles of growth factors in mouse lung neoplasia were investigated by examining receptors for platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in epithelial cell lines. Whereas nontumorigenic lung cells expressed mRNA and protein for PDGF receptor (PDGFR)-α, PDGFR-β, and EGF receptor (EGFR), five of six neoplastic lines did not. Because this exceptional tumorigenic cell line grows slowly, we hypothesized that receptor levels increased with cell stasis. To test this hypothesis, serum concentrations were manipulated, and log-phase and post-confluent cells were compared. Consistent with our hypothesis, PDGFR-α and EGFR contents, but not PDGFR-β contents, increased at stasis. Ki-ras mutation initiates lung tumorigenesis in mice, but activation of Ki-ras did not affect receptor expression. This was determined both by transfecting nontumorigenic cells with activated Ki-ras and neoplastic cells with a Ki-ras antisense construct and by diminishing Ki-ras activation by using a farnesyltransferase inhibitor. Stasis-associated upregulation of growth-factor receptor expression suggests a function in lung cell differentiation that is abrogated during neoplastic growth. Mol. Carcinog. 25:285–294, 1999. © 1999 Wiley-Liss, Inc.

Fetal mouse lung before gestation day 17 shows unique sensitivity to causation of rapidly growing tumors by N-ethylnitrosourea (ENU). Since mouse lung tumors present a mutated K-ras oncogene, we hypothesized that this special susceptibility might reflect an unusual vulnerability of the K-ras gene. Of the lung tumors caused by ENU exposure of BALB/c mice on gestation day 14, 8/25 had a codon 12 mutation in K-ras, vs 4/25 in codon 61. Of 15 tumors after day 16 exposure, three had codon 12 and four codon 61 changes. Tumors from day 18 exposure had only codon 61 mutations (11/16), all A:T to G:C changes (CGA). By contrast, codon 12 (GGT) changes included G:C to T:A, to A:T, and to C:G. These results show significant (P0.01) shift in the sensitivity of particular K-ras codons to ENU mutation, during fetal mouse lung maturation. In a test of a possible relationship to expression of K-ras, K-ras p21 was measured in lungs of fetal mice, and found to increase markedly on day 18 in comparison to days 14 and 16. Both alkylation of DNA and base damage due to reactive oxygen species are postulated as mechanisms for mutation by ENU, whose efficacies vary with state of fetal lung maturation and K-ras expression.

A state of permanent growth arrest characterises a senescent cell. Both the beneficial and deleterious effects that have accrued in senescent cells are observed in a complex organ, such as the liver. Injury to liver tissues triggers processes of regeneration and associated wound healing. Persistent injury can also lead to the neoplastic state. Recent evidence linked the senescent characteristics of the cells to the beneficial processes of wound healing and tumour surveillance in the liver. On the other hand, the secretory phenotype of senescent cells can also selectively promote undesirable neoplastic progression. In an evolutionary context, a senescent cell can function primarily as an adaptive response featuring the characteristics of altruism, trade-offs and bystander effects. Using the liver cell as a model system, this review focuses on the current knowledge of the role of senescence in these seemingly contradictory cell phenomena.

Previously we had discovered loss of DNA methylation at the DNMT3L promoter, an enzymatically-inactive DNA methyltransferase, in squamous cell carcinoma of cervix indicating association between cancer and DNMT3L. This study extends this correlation further by identifying the role of DNMT3L in nuclear reprogramming, an event central to the process of carcinogenesis. We show that in cervical cancer cell lines, overexpression of DNMT3L, which functions by regulating the activity of DNMT3A and DNMT3B, increased cellular proliferation and anchorage-independent growth. Importantly, increased DNMT3L expression resulted in changed morphology of cells but this change was gradual and observed only after several passages. Interestingly, confluent cultures of DNMT3L-overexpressing HeLa cell colonies had characteristics of iPS cells. Concomitant with the morphological changes, expression pattern of genes important in nuclear reprogramming, development and cell cycle were observed to have significantly changed. Many imprinted genes, the known targets of DNMT3L, were downregulated. The slow nature of morphological changes and genome-wide nuclear reprogramming observed upon DNMT3L overexpression reinforces its role in carcinogenesis.

Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate with demonstrated anti-proliferative and anti-cancer activity in mammary cells. We hypothesized that IP6 modulates cell cycle proteins by action on cytoplasmic signaling molecules. The effects of both pharmacological (2 mM) and physiological (100 microM) doses of IP6 on major PKC isoforms (PKCalpha, delta, epsilon, beta and zeta), PI3-K/Akt and ras/Erk1/2 were evaluated. Treatment of MCF-7 human breast cancer cells with 2 mM IP6 for 24 h caused a 3.1-fold increase in the expression of anti-proliferative PKCdelta. Similar results were observed with 100 microM IP6 at only 30-60 min post-treatment. IP6 also caused an increase in PKCdelta activity, shown by its translocation from cytosol to membrane. No changes in expression of PKC alpha, delta, epsilon, beta and zeta were detected. Additionally, IP6 caused a decrease of Erk1/2 and Akt activity. Among cell cycle control proteins, IP6 resulted in increased p27Kip1 protein levels and marked reduction of pRb phosphorylation. Specificity of the IP6 effects on p27Kip1 and pRb in MCF-7 cells (hormone-dependent) were additionally confirmed in highly invasive hormone-independent MDA-MB 231 breast cancer cells. Use of specific pharmaclogical inhibitors of PKC delta, MEK/Erk, and PI3K/Akt pathways indicated that the IP6-mediated effects on PKC delta were responsible for up-regulation of p27Kip, and pRb hypo-phosphorylation. In addition, IP6-induced apoptosis detected in MCF-7 cells appeared also to be PKC delta-dependent. Our data suggest potential usefulness of IP6 as a novel therapeutic modulator of PKC delta and p27Kip1, an important prognostic factor in human breast cancers.

BackgroundCalcineurin (CaN) is an important serine-threonine phosphatase (PP2B), which plays a crucial role in calcium-calmodulin mediated signal transduction events. Calcineurin has been implicated in pathogenesis of various diseases cardiac hypertrophy, diabetic neuropathy and Alzheimer's, however its role in neoplasia remains unclear.ResultsIn view of this we evaluated the calcineurin activity in serum and biopsy samples collected from women diagnosed with invasive squamous cell carcinoma of cervix. A significant reduction was observed in the calcineurin activity in cancer cervix patients compared to the control group. However the calcineurin activity remained unaltered in the cervical scrapes obtained from patients diagnosed with low-grade squamous intra epithelial lesions (LSIL). Interestingly the downregulation of calcineurin activity in squamous cell carcinomas was not accompanied by any significant change in DNA-binding affinity of the transcriptional factor NFAT (Nuclear Factor of Activated T-cells). All the squamous cell carcinoma samples used in the present study were positive for high-risk human papillomavirus (HPV) types.ConclusionThe present study demonstrates the downregulation of calcineurin activity in squamous cell carcinoma of cervix with high risk HPV infection. We conclude that perturbations in calcineurin-mediated pathway may be involved in development of cervical neoplasia.

Non-clear cell rat kidney tumors, inducible by N-nitroso compounds but lacking mutations in the von Hippel–Lindau (VHL) coding sequence, were examined for other VHL alterations. Neither mutations nor DNA methylation was detected in a putative promoter region. By immunohistochemistry, however, VHL protein level was evidently reduced in six of the eight eosinophilic renal epithelial tumors and in all the ten nephroblastomas. Immunoblotting of normal kidney detected two VHL proteins of 20 and 22 kDa in a 16-day-old fetal rat but only 20 kDa protein in an adult rat. This is the first demonstration of VHL alteration at the protein level.

Preconceptional exposure of male NIH Swiss mice to chromium(III) chloride resulted in increased incidence of neoplastic and non-neoplastic changes in their progeny, including lung tumors in females [Toxicol. Appl. Pharmacol. 158 (1999) 161-176]. Since mutations in the K-ras protooncogene are frequent, early changes in mouse lung tumors, we investigated possible mutational activation of this gene as a mechanism for preconceptional carcinogenesis by chromium(III). These offspring had lived until natural death at advanced ages (average 816+/-175 days for controls, 904+/-164 for progeny of chromium-treated fathers). Mutations of K-ras, analyzed by single-strand conformation polymorphism and sequencing, were, in codon 12, wild type GGT (glycine), to GAT (aspartic acid); to GTT (valine); and to CGT (arginine); and in codon 61, wild-type CAA (glutamine), to CGA (arginine). K-ras mutation frequencies in lung tumors were very similar in control progeny (4/14) and in progeny of chromium-treated fathers (5/15). Thus, germline mutation or tendency to spontaneous mutation in K-ras does not seem to be part of the mechanism of preconceptional carcinogenesis here. However, an additional interesting observation was that K-ras mutations were much more frequent in lung carcinomas (8/16) than in adenomas (1/13) (P=0.02), for all progeny combined. This was not related to age of the tumor-bearing mice or the size of the tumors. K-ras mutations may contribute to malignant tumor progression during aging, of possible relevance to the putative association of such mutations with poor prognosis of human lung adenocarcinomas.

Mutational activation of the K-ras oncogene often occurs in human and mouse lung adenocarcinomas. Since K-ras p21 functions in trans-membrane signaling, we have investigated whether the amount of this protein in lung cell membranes is a variable that could influence lung tumorigenesis, either due to genetic differences or in response to tumor promoters. The six mouse strains assessed showed little difference in the total lung K-ras p21 after immunoprecipitation and immunoblotting. However, amount of ras p21 in the membrane fraction showed significant differences, with C57BL/6 and BALB/c having 3-5-fold more than NIH Swiss, AKR and DBA mice. Interestingly, a congenic AKR strain having the Ahr b-1 Ah receptor allele from C57BL/6 mice (designated AKR.B6Ah) had high lung membrane K-ras p21 similar to that of C57BL/6. To test for possible changes related to lung tumor promotion, mice were treated with a promotional dose of TCDD (5 nmol/kg). After 48 h C57BL/6 lungs showed an increase in p21 in both total and membrane fractions. BALB/c, DBA and Swiss mice showed an increase only in membranes. There was no change in the AKR and AKR.B6Ah. Aroclor 1254 (250 mg/kg) caused an increase in membrane/cytosol ratio in Swiss mice. Thus the membrane :cytosol K-ras p21 ratio may be influenced by the Ahr phenotype, and TCDD and PCBs can induce p21 or increase its membrane level in certain strains, but these properties are not fully dependent on Ahr receptor type. In confirmation of the relevance of these findings for the tumor target cell type, the immortalized alveolar type 2 E10 cell line presented K-ras p21 in membrane, and this was increased 4-fold by treatment with 10 nM TCDD.

Recent studies point to a close connection between cancer and ageing. Cellular senescence when initiated early in cancerous cells results in growth suppression due to permanent growth arrest. Sirtuins, belonging to family of the NAD-dependent deacetylases, play a crucial role in cellular metabolism and organismal ageing/ longevity. However, the role of human Sirtuin isoforms in malignancies and cellular senescence is still not clearly discernible. The present study is therefore designed to evaluate the correlation of SIRT1 expression with proliferation marker Ki-67, and growth arrest/senescence marker p27, during cervical cancer progression. The expression was evaluated by immunohistochemistry in 70 formalin fixed archival human cervical samples: normal/ASCUS (N= 20), squamous intraepithelial lesions (SIL N=20) and invasive squamous cell carcinoma (SCC, N= 30 samples). All the intraepithelial lesions and SCC cases were positive for human papilloma virus as detected by in-situ hybridization. The SIRT1 expression was either absent or feeble in cytoplasm of normal/ASCUS cervical epithelium, while a progressive increase in its expression was noted in 65% of preneoplastic lesions (SIL). Intriguingly, the invasive carcinoma showed heterogeneous pattern for both expression and localization of SIRT1: ranging from negative to strong expression levels and mixed localization pattern in cytoplasm only or both in nucleus and cytoplasm. The growth arrest/senescence marker p27 showed elevated level of expression in SIL cases compared to SCC (75%). Interestingly, the increased nucleo-cytoplasmic expression pattern of SIRT1 correlated well with the increased expression of p27 in the intraepithelial preneoplastic lesion. In conclusion, our results suggest activation of growth suppressive pathways by SIRT1-p27 regulation, atleast in the in preneoplastic lesions of cancer cervix which may help retard its progression to invasive cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3951. doi:1538-7445.AM2012-3951