Results of the baseline positron emission tomography can customize therapy of localized esophageal adenocarcinoma patients who achieve a clinical complete response after chemoradiation.

BACKGROUND: Patients with localized esophageal adenocarcinoma (EAC) who achieve a clinical complete response (clinCR) after preoperative chemoradiation (trimodality therapy; TMT) or definitive chemoradiation (bimodality therapy; BMT) live longer than those who achieve a

Selective toxicity of MKT-077 to cancer cells is mediated by its binding to the hsp70 family protein mot-2 and reactivation of p53 function.

MKT-077, a cationic rhodacyanine dye analogue has been under preclinical cancer therapeutical trials because of its selective toxicity to cancer cells. Its cellular targets and mechanism of action remain poorly understood. Here we report that MKT-077 binds to an hsp70 family member, mortalin (mot-2), and abrogates its interactions with the tumor suppressor protein, p53. In cancer cells, but not in normal cells, MKT-077 induced release of wild-type p53 from cytoplasmically sequestered p53-mot-2 complexes and rescued its transcriptional activation function. Thus, MKT-077 may be particularly useful for therapy of cancers with wild-type p53.

A highly expressed 81 kDa protein in immortalized mouse fibroblast: its proliferative function and identity with ezrin.

An 81 kDa protein was found to be highly expressed in spontaneously immortalized CD1-ICR mouse fibroblasts, RS-4, as compared to normal fibroblasts. RS-4 cells have a reduced serum requirement and exhibit multilayered growth in vitro but are not tumorigenic. The protein was purified from RS-4 cell extracts and used to obtain a polyclonal antibody that specifically immunoprecipitated an 81 kDa protein from cell lysates. Immunocloning of its cDNA and sequence analysis revealed its identity with ezrin, an F-actin binding protein that is a component of the cortical cytoskeleton. Microinjection of the purified IgG fraction of the anti-p81 antiserum into the cytoplasm of RS-4 cells blocked their entry into S phase suggesting that the protein has a proliferative function. Immunostaining of normal mouse tissues showed that the expression of p81/ezrin was highest in proliferating cell populations. Unlike RS-4, NIH3T3 cells exhibit contact inhibition and express levels of p81/ezrin similar to those of normal fibroblasts. When NIH3T3 cells were transfected with p81/ezrin cDNA they lost contact inhibition and thus resembled RS-4 cells. The study demonstrates a proliferative function of p81/ezrin and suggests its involvement in pathways that negatively regulate contact inhibition.

Gros1, a potential growth suppressor on chromosome 1: its identity to basement membrane-associated proteoglycan, leprecan.

By immunoscreening with an antibody raised against a plasma membrane protein, we have cloned a growth suppressor gene, Gros1 and assigned it to short arm of human chromosome 1. Two alternatively spliced forms of the gene encoding 84- and 41-kDa (carboxy-terminus truncated) proteins were cloned. The two transcripts, 4.4 and 2.7 kb, were expressed weakly in most of the human tissues, with a high expression of the smaller transcript in placenta, ovary and testis. Normal human fibroblasts in culture showed two transcripts, with a higher level of expression of the 4.4 kb transcript. Transformed cells on the other hand showed predominant expression of the 2.7 kb transcript. Two Gros1 transcripts were also detected in most of the mouse tissues. Stable transfection of the mouse cDNA encoding the 85-kDa protein into NIH3T3 cells resulted in their slow growth and reduced colony-forming efficiency. Stable clones expressing antisense RNA on the other hand exhibited higher colony forming efficiency. While our data implied that Gros1 is a novel growth suppressor gene on human chromosome 1, an independent study has recently characterized its rat-homolog as a leucine proline-enriched novel basement membrane-associated proteoglycan leprecan. We describe here cloning, expression and biological activity analysis implying that this novel proteoglycan is a potential growth suppressor on chromosome 1p31, frequently altered in many malignancies.

Malignant transformation of NIH3T3 cells by overexpression of mot-2 protein.

The murine mortalin genes, mot-1 and mot-2, are members of the hsp70 family of proteins and differ from each other by only two amino acid residues. Mot-1 is expressed in normal cells and has pancytosolic cellular distribution whereas mot-2 is found in the perinuclear region of immortal cells. We report here that a high level of expression of mot-2 protein resulted in malignant transformation of cells as analysed by anchorage independent growth and nude mice assays. A high level of protein expression is attributed to the 900 bp 3' untranslated region of the cDNA which does not have any transforming activity per se. Mortalin cDNA clones isolated from human transformed cells were also found to have transforming activity in similar assays and a high level of expression was apparent in some of the human immortalized cells that showed non-pancytosolic mortalin immunofluorescence. Taken together, the data suggest that nonpancytosolic mortalin may have a role in tumorigenesis.

Inhibitors of cGMP-dependent protein kinase block senescence induced by inactivation of T antigen in SV40-transformed immortal human fibroblasts.

Immortal human fibroblasts isolated following transfection with thermolabile simian virus 40 T antigen lost division potential upon shift up in temperature due to heat inactivation of the antigen. Such cells showed a concomitant change in the distribution of a mortality marker, mortalin, from a juxtanuclear cap like distribution of immortal cells to a uniform cytosolic distribution of mortal cells. We made an attempt to modulate the above inducible system of cellular senescence using various protein kinase inhibitors. Among the indolocarbazole type inhibitors tested, only KT5823, defined as a specific inhibitor of cGMP-dependent protein kinase, blocked the loss of division potential as determined by cell growth and colony forming ability. This inhibitor also prevented the above change in mortalin distribution due to temperature shift. In addition, the isoquinoline sulfonamide derivatives H8, H9, H88 and H89, all shown to inhibit cGMP-dependent protein kinase, suppressed the senescence. Inhibitors specific to other types of protein kinases, protein phosphatases or tyrosine kinases tested had no effect. Since there was no difference between the effective and non-effective inhibitors in their effects on cell cycle progression, cell cycle arrest by itself cannot account for the above phenomenon. These results suggest that a signaling pathway possibly mediated by cGMP-dependent protein kinase is involved in the induction of cellular senescence.

Enhanced expression of multiple forms of VEGF is associated with spontaneous immortalization of murine fibroblasts.

The mechanism(s) involved in immortalization that constitute the first step during malignant transformation has been the subject of our interest. By the use of spontaneously immortalized mouse embryonic fibroblasts we have earlier identified two stages of immortalization which are characterized by growth characteristics of the cells, their conditioned medium and the protein markers such as p53, p81 and mortalin (Kaul et al. (1994) Biochim. Biophys. Acta, in press). The present study was planned to purify the mitogenic factors from the conditioned medium of stage II cells. Sequential purification by chromatography followed by peptide sequencing has characterized one of these as vascular endothelial growth factor (VEGF). Further analysis by RT-PCR suggests that the spontaneously immortalized stage II fibroblasts have enhanced synthesis and secretion of VEGF as compared to their mortal parent cells. Expression of a novel 304 bp long form of VEGF is identified in immortal fibroblasts in addition to the three known alternatively spliced forms. The study points to the involvement of VEGF function during spontaneous immortalization of mouse embryonic fibroblasts.

Identification of genetic events involved in early steps of immortalization of mouse fibroblasts.

The spontaneously immortalized early passaged fibroblasts from three different strains of mouse are observed to represent two distinct stages of immortalization. The cells at stage I are characterized by slow growth rate, contact inhibition and requisition of serum factors for their growth and proliferation. Stage II cells are marked by fast, multilayer growth that is independent of serum supplementation in growth medium and by the elevated levels of the two marker proteins, i.e., p53 and p81. The change from cytosolic distribution of mortalin, a senescence inducing protein (J. Biol. Chem. (1993) 268, 6615-6621; 22239-22242) to the perinuclear locale is detected as an early event during cellular immortalization. Furthermore, the distinct stages could be characterized by thermal analysis of intact cells, that to the best of our knowledge is employed for the first time for the analysis of cellular mortal and immortal phenotypes. The study characterizes at least two distinct end points in rodent transformation suggesting that there are multiple routes to immortalization.

Evaluation of the anti-genotoxicity of leaf extract of Ashwagandha.

We have undertaken the studies to investigate the presence of various activities of the leaf extract of Ashwagandha (Lash), a commonly used shrub in Indian traditional medicine, Ayurveda. In the present study, we studied the effect of Lash against MNNG-induced genotoxicity in onion root tip cells. We report that Lash offered substantial protection against the mutagenic effects of MNNG.

Study of the Risk Factors for Cesarean Delivery in Induced Labors at Term.

BACKGROUND: Induction of labor has become one of the most common interventions in obstetrics. Induction is indicated when the benefits to either mother or fetus outweigh those of continuing the pregnancy. Maternal complication rates that are increased in association with labor induction include cesarean delivery, chorioamnionitis, abruptio placentae, and uterine atony. So identifying those pregnancies that can be induced with low risk of cesarean delivery is important. The aim of this study was to identify those factors which influence the risk of emergency cesarean delivery in induced labors at term. METHODS: It is a case-control study conducted at Tertiary care center and affiliated hospitals. In this study, odds ratio with 95 % confidence interval is taken as a measure of relative risk. Patients were evaluated for risk factors for cesarean section using logistic regression. Cases include all women who were induced at term and delivered by emergency cesarean section. Controls include all women who were induced at term and delivered vaginally. RESULTS: The risk factors for cesarean delivery are advanced maternal age at delivery (≥35 years), high early pregnancy body mass index (≥30 kg/m(2)), nulliparity, low preinduction Bishops score (<5), gestational diabetes mellitus, and intrauterine growth restriction. CONCLUSION: Women with multiple risk factors for cesarean can be taken up for elective cesarean section rather than inducing them at term.

Ramucirumab for the treatment of gastroesophageal cancers.

INTRODUCTION: In 2014, the U.S. Food and Drug Administration (FDA) approved ramucirumab for use in the second line setting of advanced or metastatic, gastric or gastroesophageal adenocarcinoma (GEAC) based on the result of Phase III clinical trials; REGARD and RAINBOW. AREAS COVERED: We briefly review the mechanisms of angiogenesis, anti-angiogenic therapy, and current status of advanced GEAC treatment then highlight the challenges and future prospects of novel molecular targeted agents. EXPERT OPINION: Although both the REGARD and RAINBOW trials met their primary endpoints of significantly prolonged overall survival (OS) and progression-free survival (PFS), the magnitude of the difference is still relatively modest. Given that ramucirumab alone has a marginal effect, a combination of paclitaxel and ramucirumab is strongly preferred as a second line therapy. To maximize the impact of ramucirumab in patients with GEAC, we can leverage the recent pharmacokinetics (PK) data of ramucirumab from the REGARD and RAINBOW trials. In addition, the quest for identifying biomarkers to select patients who are likely to benefit the most should continue. It is our firm belief that taxanes should no longer be added to the frontline regimens in most cases, given the success of the taxane/ramucirumab in the second line setting.

An N-terminal region of mot-2 binds to p53 in vitro.

The mouse mot-2 protein was earlier shown to bind to the tumor suppressor protein, p53. The mot-2 binding site of p53 was mapped to C-terminal amino acid residues 312-352, which includes the cytoplasmic sequestration domain. In the present study, we have found that both mot-1 and mot-2 bind to p53 in vitro. By using His-tagged deletion mutant proteins, the p53-binding domain of mot-2 was mapped to its N-terminal amino acid residues 253-282, which are identical in mot-1 and mot-2 proteins. Some peptides containing the p53-binding region of mot-2 were able to compete with the full-length protein for p53 binding. The data provided rationale for in vitro binding of mot-1 and mot-2 proteins to p53 and supported the conclusion that inability of mot-1 protein to bind p53 in vivo depends on secondary structure or its binding to other cellular factors. Most interestingly, the p53-binding region of mot-2 was common to its MKT-077, a cationic dye that exhibits antitumor activity, binding region. Therefore it is most likely that MKT-077-induced nuclear translocation and restoration of wild-type p53 function in transformed cells takes place by a competitional mechanism.

Structurally and functionally distinct mouse hsp70 family members Mot-1 and Mot-2 proteins are encoded by two alleles.

The mouse mortalin proteins Mot-1 and Mot-2 differ by two amino acids in their carboxy-terminus. These proteins are differentially localized in the cell cytoplasm and have contrasting biological activities. The genetic relationship between Mot-1 and Mot-2 was deciphered by mouse family analyses. Mot-1 and Mot-2 segregated in F1 and F2 progeny, providing direct evidence that the two proteins are encoded by two alleles.

Identification and differential expression of yeast SEC23-related gene (Msec23) in mouse tissues.

We have isolated a yeast SEC23-related clone (Msec23) from mouse fibroblast cDNA library. It has an open reading frame of 1721 bp (64% homologous to SEC23-2.3 kb) which can potentially encode a 64.7 kDa protein (61% homologous to 85.4 kDa product of SEC23). The deduced Msec23 protein (Msec23p) sequence contains three successive Ig-like domains at the N-terminus followed by amphipathic alpha-helical regions, suggesting the potential of Msec23p to interact with other protein components. Further, Msec23 is differentially expressed in mouse tissues with its high level in brain and fibroblasts.

Mouse and human chromosomal assignments of mortalin, a novel member of the murine hsp70 family of proteins.

Mortalin has been shown to exhibit differential distributions in cells with mortal and immortal phenotypes. In the present study, we report mot-2 cDNA cloning from RS-4 cells--an immortal clone from CD1-ICR mouse embryonic fibroblasts--and the chromosomal assignments of mortalin related genes to mouse chromosomes 18 and X by fluorescence in situ hybridization. Similar analysis assigned the gene to chromosome 5q31.1 in human.

Effect of butylated hydroxytoluene on the life span of Drosophila bipectinata.

The median and the maximum life spans of Drosophila bipectinata increased after feeding with various concentrations (10 microM, 10(2) microM and 10(3) microM) of butylated hydroxytoluene (BHT). Insects fed on the diet supplemented with 10(3) microM BHT had decreased rate of lipid peroxidation (measured by thiobarbituric acid test) with respect to the controls. It is suggested that the antioxidant BHT, which scavenges free radicals, prolongs the life span of Drosophila.

Studies on catalase in ageing Zaprionus paravittiger (Diptera) with special reference to an antioxidant feeding.

Zaprionus paravittiger fed with an antioxidant (sodium hypophosphite, 1 X 10(3) microM) supplemented diet exhibited adaptive compensatory responses in catalase activity (quantitative as well as qualitative). The longevity and catalase activity were found to be positively linked. The study denotes that free radical formation and antioxygenic defenses are closely associated and are the possible determinants of life span and ageing.

An antioxidant induced alterations in peroxidase activity in ageing Zaprionus paravittiger (Diptera).

Sodium hypophosphite (1 x 10(3) microM) supplementation in the diet of Zaprionus paravittiger resulted in adaptive responses in the quantitative as well as qualitative activity of peroxidase. The changes in peroxidase were in compliance with the earlier reported sodium hypophosphite (SHP)-induced prolonged life span of flies (Wadhwa, R., Arch. Gerontol. Geriatr., 6 (1987) 141-147) and support the concept that antioxidative enzymes may serve as important longevity determinants.

NIH 3T3 cells malignantly transformed by mot-2 show inactivation and cytoplasmic sequestration of the p53 protein.

In previous studies we have reported that a high level of expression of mot-2 protein results in malignant transformation of NIH 3T3 cells as analyzed by anchorage independent growth and nude mice assays [Kaul et al., Oncogene, 17, 907-11, 1998]. Mot-2 was found to interact with tumor suppressor protein p53. The transient overexpression of mot-2 was inhibitory to transcriptional activation function of p53 [Wadhwa et al., J. Biol. Chem., 273, 29586-91, 1998]. We demonstrate here that mot-2 transfected stable clone of NIH 3T3 that showed malignant properties indeed show inactivation of p53 function as assayed by exogenous p53 dependent reporter. The expression level of p53 in response to UV-irradiation was lower in NIH 3T3/mot-2 as compared to NIH 3T3 cells and also exhibited delay in reaching peak. Furthermore, upon serum starvation p53 was seen to translocate to the nucleus in NIH 3T3, but not in its mot-2 derivative. The data suggests that mot-2 mediated cytoplasmic sequestration and inactivation of p53 may operate, at least in part, for malignant phenotype of NIH 3T3/mot-2 cells. NIH 3T3/mot-2 cells show inactivation of p53 protein.