Workflow in transgenic mice for the study of specific cancers associated with the post-menopausal state.

Beta-hCG is associated with poor patient prognosis in several cancers, but the particular pathophysiology of beta-hCG in post-menopausal women remains unaddressed. Specific steps to culture Lewis lung carcinoma (LLC1) tumor cells are enumerated. Ovariectomy of syngeneic, beta-hCG transgenic mice is discussed, employing a protocol designed to ensure high survival. Implantation of LLC1 tumor cells in these mice is also described. The workflow can be easily adapted in the study of other cancers associated with the post-menopausal stratum. For complete details on the use and execution of this protocol, please refer to Sarkar et al. (2022).1.

Progesterone limits the tumor-promoting effects of the beta-subunit of human chorionic gonadotropin via non-nuclear receptors.

The post-menopausal state in women is associated with increased cancer incidence, the reasons for which remain obscure. Curiously, increased circulating levels of beta-hCG (human chorionic gonadotropin) (a hormonal subunit linked with tumors of several lineages) are also often observed post-menopause. This study describes a previously unidentified interplay between beta-hCG and progesterone in tumorigenesis. Progesterone mediated apoptosis in beta-hCG responsive tumor cells via non-nuclear receptors. The transgenic expression of beta-hCG, particularly in the absence of the ovaries (a mimic of the post-menopausal state) constituted a potent pro-tumorigenic signal. Significantly, the administration of progesterone had significant anti-tumor effects. RNA-seq profiling identified molecular signatures associated with these processes. TCGA analysis revealed correlates between the expression of several newly identified genes and poor prognosis in post-menopausal patients of lung adenocarcinoma, colon adenocarcinoma, and glioblastoma. Specifically in these women, the detection of intra-tumoral/extra-tumoral beta-hCG may serve as a useful prognostic indicator, and treatment with progesterone on its detection may prove beneficial.

CNOT3 interacts with the Aurora B and MAPK/ERK kinases to promote survival of differentiating mesendodermal progenitor cells.

Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B that requires sequences in the NOT box domain of CNOT3 and regulates MAPK/ERK signaling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse embryonic stem cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of cells with ectodermal characteristics. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4, and Wnt3 due to reduced survival and proliferation of differentiating mesendoderm cells. We also show that the double mutation alters the balance of interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2. Our results identify a potential adaptor function for CNOT3 that regulates the Ras/MEK/ERK pathway during embryogenesis.

Cloning and immunobiochemical analyses on recombinant chymopapain allergen Cari p 2 showing pollen-fruit cross-reaction.

Papaya is reported to trigger food and respiratory allergy. Here, we identified chymopapain Cari p 2 as an allergen that can sensitize atopic individuals through fruit consumption followed by respiratory hazards through pollen exposure. Recombinant Cari p 2 displayed IgE-reactivity with 78% of papaya allergic sera. rCari p 2 also displayed allergenic activity through basophil degranulation. rCari p 2 is correctly folded and showed irreversible denaturation in the melting curve. rCari p 2 displayed IgE-cross-reactivity with homologous cysteine proteases from kiwi and pineapple. Cari p 2 transcript was also detected in papaya pulps. rCari p 2 was resistant to pepsin digestion and retained IgE-reactivity after 60 minutes of pepsin digestion. In mouse model, rCari p 2 was found to elicit inflammatory responses in the lung and gastrointestinal epithelium. Hence, Cari p 2 is a newly characterized allergen with diagnostic and immunotherapeutic potential for managing allergic disorders in papaya sensitized individuals.

The transgenic expression of the ß-subunit of human chorionic gonadotropin influences the growth of implanted tumor cells.

The beta subunit of human chorionic gonadotropin (ßhCG) is secreted by various tumors, and its presence associated with poor prognosis. Though exogenous hCG elicits the synthesis of molecules associated with angiogenesis, invasion, immune suppression and chemoresistance from responsive tumor cells in vitro, the influence of cell-extrinsic ßhCG on tumorigenesis in vivo has not been adequately explored. Female C57BL/6-/- × FVBßhCG/- F1 transgenic mice demonstrated ovarian hyperplasia and pituitary adenomas; transcripts of hCG-driven, tumor-associated molecules were heightened in the pituitary. Upon the implantation of Lewis Lung Carcinoma cells (murine lung tumor cells derived from C57BL/6 mice) in transgenic mice, tumor incidence and volume were enhanced, and increased transcription and expression of hCG-driven, tumor-associated molecules was observed in excised tumors. While treatment of these mice with Cabergoline (a potent dopamine receptor agonist) had no significant effects, ovariectomy resulted in a reduction in the lag phase, accompanied by an increase in tumor incidence and volume upon Lewis Lung Carcinoma cell implantation. In tumors derived from Lewis Lung Carcinoma cell-implanted ovariectomized, transgenic mice, the transcription and expression of hCG-driven, tumor-associated molecules remained elevated and enhanced animal mortality was observed. Cell-extrinsic ßhCG can therefore induce pro-tumorigenic effects in vivo (even on tumor lineages not part of the reproductive axis), with ovarian products mediating an ameliorating influence.

DEAD box RNA helicases: crucial regulators of gene expression and oncogenesis.

DEAD box protein family of RNA helicases are vital players of RNA metabolism, and constitute the largest family of RNA helicases. Members of this family share nine conserved motifs including an Asp-Glu-Ala-Asp motif, giving this family its characteristic name as DEAD box RNA helicases. These conserved motifs confer RNA binding and RNA unwinding properties. Besides functioning in RNA metabolism, emerging evidences suggests several DEAD box RNA helicases to possess potential roles in regulating gene expression by acting as a transcriptional co-activator. Many of them are deregulated in cancers, and are implicated in possessing oncogenic potential. On the contrary, each of them also possesses tumor suppressive property in a context dependent manner. In this review, we discuss the mechanistic insights of gene regulation by DEAD box RNA helicases, and their significance in cancers.

Exosome-mediated delivery of the intrinsic C-terminus domain of PTEN protects it from proteasomal degradation and ablates tumorigenesis.

PTEN mutation is a frequent feature across a plethora of human cancers, the hot-spot being its C-terminus (PTEN-CT) regulatory domain resulting in a much diminished protein expression. In this study, the presence of C-terminus mutations was confirmed through sequencing of different human tumor samples. The kinase CKII-mediated phosphorylation of PTEN at these sites makes it a loopy structure competing with the E3 ligases for binding to its lipid anchoring C2 domain. Accordingly, it was found that PTEN-CT expressing stable cell lines could inhibit tumorigenesis in syngenic breast tumor models. Therefore, we designed a novel exosome-mediated delivery of the intrinsic PTEN domain, PTEN-CT into different cancer cells and observed reduced proliferation, migration, and colony forming ability. The delivery of exosome containing PTEN-CT to breast tumor mice model was found to result in significant regression in tumor size with the tumor sections showing increased apoptosis. Here, we also report for the first time an active PTEN when its C2 domain is bound by PTEN-CT, probably rendering its anti-tumorigenic activities through the protein phosphatase activity. Therefore, therapeutic interventions that focus on PTEN E3 ligase inhibition through exosome-mediated PTEN-CT delivery can be a probable route in treating cancers with low PTEN expression.

DEAD-box protein p68 is regulated by ß-catenin/transcription factor 4 to maintain a positive feedback loop in control of breast cancer progression.

INTRODUCTION: Nuclear accumulation of ß-catenin is important for cancer development and it is found to overlap with p68 (DDX5) immunoreactivity in most breast cancers, as indicated by both clinical investigations and studies in cell lines. In this study, we aim to investigate the regulation of p68 gene expression through ß-catenin/transcription factor 4 (TCF4) signaling in breast cancer. METHODS: Formalin-fixed paraffin-embedded sections derived from normal human breast and breast cancer samples were used for immunohistochemical analysis. Protein and mRNA expressions were determined by immunoblotting and quantitative RT-PCR respectively. Promoter activity of p68 was checked using luciferase assay. Occupancy of several factors on the p68 promoter was evaluated using chromatin immunoprecipitation. Finally, a syngeneic mouse model of breast cancer was used to assess physiological significance. RESULTS: We demonstrated that ß-catenin can directly induce transcription of p68 promoter or indirectly through regulation of c-Myc in both human and mouse breast cancer cells. Moreover, by chromatin immunoprecipitation assay, we have found that both ß-catenin and TCF4 occupy the endogenous p68 promoter, which is further enhanced by Wnt signaling. Furthermore, we have also established a positive feedback regulation for the expression of TCF4 by p68. To the best of our knowledge, this is the first report on ß-catenin/TCF4-mediated p68 gene regulation, which plays an important role in epithelial to mesenchymal transition, as shown in vitro in breast cancer cell lines and in vivo in an animal breast tumour model. CONCLUSIONS: Our findings indicate that Wnt/ß-catenin signaling plays an important role in breast cancer progression through p68 upregulation.

Risk factors for and epidemiology of community-onset vancomycin-resistant Enterococcus faecalis in southeast Michigan.

BACKGROUND: Given the known link between vancomycin-resistant Enterococcus faecalis (VREF) and vancomycin-resistant Staphylococcus aureus (VRSA), the recent increase in prevalence of VREF in southeast Michigan has raised concerns about the presence of a large "community" reservoir of VREF. Efforts to control its spread face some important challenges. METHODS: Patients with clinical isolates of community-onset (CO) VREF (cases) were compared with matched uninfected controls (study 1) and patients with hospital-onset (HO) VREF (study 2). Here, CO was defined as a hospital stay of ≤2 days before VRE isolation. RESULTS: Independent predictors for the isolation of CO-VREF compared with uninfected controls were nonhome residence; chronic skin ulcers; previous invasive procedures/surgery; exposure to cephalosporin, penicillin, and/or vancomycin; immunosuppressive status; and the presence of indwelling devices. Independent predictors for isolation of CO-VREF compared with HO-VREF included no stay in an intensive care unit in the previous 3 months and recent hospitalization. VREF isolation from wounds and aminoglycoside exposure were inversely associated with isolation of CO-VREF. CONCLUSIONS: Health care-related exposures and antimicrobial exposures are risk factors for the isolation of CO-VREF. Regional infection control practices are imperative in controlling CO-VREF, in addition to the emergence and spread of VRSA.

2,2'-diphenyl-3,3'-diindolylmethane: a potent compound induces apoptosis in breast cancer cells by inhibiting EGFR pathway.

Despite recent advances in medicine, 30-40% of patients with breast cancer show recurrence underscoring the need for improved effective therapy. In this study, by in vitro screening we have selected a novel synthetic indole derivative 2,2'-diphenyl-3,3'-diindolylmethane (DPDIM) as a potential anti- breast cancer agent. DPDIM induces apoptosis both in vitro in breast cancer cells MCF7, MDA-MB 231 and MDA-MB 468 and in vivo in 7,12-dimethylbenz[α]anthracene (DMBA) induced Sprague-Dawley (SD) rat mammary tumor. Our in vitro studies show that DPDIM exerts apoptotic effect by negatively regulating the activity of EGFR and its downstream molecules like STAT3, AKT and ERK1/2 which are involved in the proliferation and survival of these cancer cells. In silico predictions also suggest that DPDIM may bind to EGFR at its ATP binding site. DPDIM furthermore inhibits EGF induced increased cell viability. We have also shown decreased expression of pro-survival factor Bcl-XL as well as increase in the level of pro-apoptotic proteins like Bax, Bad, Bim in DPDIM treated cells in vitro and in vivo. Our results further indicate that the DPDIM induced apoptosis is mediated through mitochondrial apoptotic pathway involving the caspase-cascade. To the best of our knowledge this is the first report of DPDIM for its anticancer activity. Altogether this report suggests that DPDIM could be an effective therapeutic agent for breast cancer.

The maternal drinking history guide: development of a national educational tool.

BACKGROUND: The National Taskforce for the development of screening tools for FASD has identified maternal drinking as a critical area that should be screened. We describe the steps of development and implementation of a knowledge translation program for health care providers. The slide presentation is attached in English and French to allow its maximal use. METHODS: In 2010, the National Taskforce for the development of screening tools for FASD identified maternal drinking as a critical area that should be screened. The systematic review and associated recommendations have been published and were included in the toolkit developed by the Canadian Association of Paediatric Health Centres with funding support from the Public Health Agency of Canada. Effective inquiry of maternal drinking can be conducted at three levels: Primary level, as part of practice-based screening; Level 2 use of structured questionnaires; and Level 3 laboratory-based screening. CONCLUSION: It was acknowledged that most physicians do not ask women of reproductive age questions regarding their drinking habits, and the Taskforce was seriously concerned that even an effective guide may not change practice at the primary level. To that end, the Taskforce developed a three phase Knowledge Translation plan, to ensure that the educational program developed will be optimally effective for Canadian healthcare providers.

Folate Intake, MTHFR Polymorphisms, and the Risk of Colorectal Cancer: A Systematic Review and Meta-Analysis.

Background. The objective was to determine whether relationships exist between the methylene-tetrahydrofolate reductase (MTHFR) polymorphisms and risk of colorectal cancer (CRC) and examine whether the risk is modified by level of folate intake. Methods. MEDLINE, Embase, and SCOPUS were searched to May 2012 using the terms "folic acid," "folate," "colorectal cancer," "methylenetetrahydrofolate reductase," "MTHFR." Observational studies were included which (1) assessed the risk of CRC for each polymorphism and/or (2) had defined levels of folate intake for each polymorphism and assessed the risk of CRC. Results. From 910 references, 67 studies met our criteria; hand searching yielded 10 studies. The summary risk estimate comparing the 677CT versus CC genotype was 1.02 (95% CI 0.95-1.10) and for 677TT versus CC was 0.88 (95% CI 0.80-0.96) both with heterogeneity. The summary risk estimates for A1298C polymorphisms suggested no reduced risk. The summary risk estimate for high versus low total folate for the 677CC genotype was 0.70 (95% CI 0.56-0.89) and the 677TT genotype 0.63 (95% CI 0.41-0.97). Conclusion. These results suggest that the 677TT genotype is associated with a reduced risk of developing CRC, under conditions of high total folate intake, and this associated risk remains reduced for both MTHFR 677 CC and TT genotypes.

Mechanism of ß-catenin-mediated transcriptional regulation of epidermal growth factor receptor expression in glycogen synthase kinase 3 ß-inactivated prostate cancer cells.

Wnt/ß-catenin and EGFR pathways are important in cancer development and often aberrantly activated in human cancer. However, it is very important to understand the mechanism responsible for this activation and the relation between them. Here, we report the mechanism of EGFR expression by transcriptionally active ß-catenin in GSK3ß-inactivated prostate cancer cells that eventually leads to its enhanced proliferation and survival. Expressions of ß-catenin and EGFR are elevated in various cancers specifically in prostate cancer cells, DU145. When GSK3ß is inactivated in these cells, ß-catenin gets stabilized, phosphorylated at Ser-552 by protein kinase A, accumulates in the nucleus, and regulates the expression of its target genes that include EGFR. Chromatin immunoprecipitation (ChIP) and promoter analysis revealed that the EGFR promoter gets occupied by transcriptionally active ß-catenin when elevated in GSK3ß-inactivated cells. This phenomenon not only leads to increased expression of EGFR but also initiates the activation of its downstream molecules such as ERK1/2 and Stat3, ultimately resulting in up-regulation of multiple genes involved in cell proliferation and survival.

Folate intake and the risk of colorectal cancer: a systematic review and meta-analysis.

INTRODUCTION: Folic acid fortification and supplementation to prevent neural tube defects has led to concerns regarding increased risk of colorectal cancer. The results of existing studies have been inconclusive. The purpose was to examine the relationship between level of folate intake and the incidence of colorectal cancer. METHODS: A systematic review and meta analysis were conducted. MEDLINE, Embase, and SCOPUS were searched from inception to October 2009 with the following search terms "folic acid," "folate", "colorectal cancer," "colon neoplasms," rectal neoplasms." Observational studies in adult populations were included that defined levels of folate intake and incidence of colorectal cancer. RESULT: Out of 6427 references, 27 studies met our inclusion criteria. The summary risk estimate for case control studies comparing high versus low total folate intake was 0.85 (CI 95% 0.74-0.99) with no significant heterogeneity among studies. Similarly, for cohort studies, the resulting summary risk estimate for high versus low dietary folate intake was 0.92 (CI 95% 0.81-1.05) with no significant heterogeneity. However, defining what represents a higher intake of folic acid is difficult as there is variability in the upper limit of folic acid intake used in the studies. DISCUSSION: These results suggest that higher folate intake levels offer a reduction in one of the perceived risks associated with developing colorectal cancer. These data can serve to help reassure women planning a pregnancy to increase folic intake during the preconception period to levels sufficient to prevent neural tube defects.

Screening and recording of alcohol use among women of child-bearing age and pregnant women.

A woman's alcohol use during pregnancy is one of the top preventable causes of birth defects and developmental disabilities that are known as fetal alcohol spectrum disorders (FASD). The social and economic burden of FASD is substantial. Lifetime direct tangible costs per individual related to health care, education and social services in Canada have been estimated to be $1.4 million. Screening women of child-bearing age and pregnant women and recording their alcohol consumption is a practical process to identify and evaluate women at-risk and to identify potentially exposed infants. The FASD Advisory Workgroup proposes the following three levels of screenings which should be done on consenting women: Level I screening involves practice-based approaches that can be used by health care providers when talking to women about alcohol use, such as motivational interviewing and supportive dialogue. Level II screening includes a number of structured questionnaires that can be used with direct questioning (TLFB) or indirect /masked screening (AUDIT, BMAST / SMAST, CAGE, CRAFFT, T-ACE, TWEAK). Level III screening includes laboratory-based tools that can be used to confirm the presence of a drug, its level of exposure and determine the presence of multiple drugs. There are challenges and limitations in the use of the screening and assessment tools outlined. For example, the single question about alcohol use and the various questionnaires rely on a woman to provide details about her alcohol use. There is no consensus on the appropriate screening to use across Canada as each provincial / territorial jurisdiction, health care organization and healthcare provider uses a variety of formal and informal screening tool. In addition, there are inconsistent processes across Canada for the recording of the alcohol use in a woman's chart and the transfer of the information to the infant and the child's health records. The FASD Advisory Workgroup proposes eleven recommendations to improve the screening and recording processes for alcohol use in women of child-bearing age and pregnant women.

Pregnancy outcome following gestational exposure to azithromycin.

BACKGROUND: Azithromycin is an azalide antibiotic with an extensive range of indications and has become a common treatment option due to its convenient dosing regimen and therapeutic advantages. Human studies addressing gestational use of azithromycin have primarily focused on antibiotic efficacy rather than fetal safety. Our primary objective was to evaluate the possibility of teratogenic risk following gestational exposure to azithromycin. METHODS: There were 3 groups of pregnant women enrolled in our study: 1) women who took azithromycin, 2) women exposed to non-teratogenic antibiotics for similar indications, and 3) women exposed to non-teratogenic agents. They were matched for gestational age at time of call, maternal age, cigarette and alcohol consumption. Rates of major malformations and other endpoints of interest were compared among the three groups. RESULTS: Pregnancy outcome of 123 women in each group was ascertained. There were no statistically significant differences among the three groups in the rates of major malformations; 3.4% (exposed) versus 2.3% (disease matched) and 3.4% (non teratogen) or any other endpoints that were examined. In the azithromycin group, 88 (71.6%) women took the drug during the first trimester CONCLUSION: Results suggest that gestational exposure to azithromycin is not associated with an increase in the rate of major malformations above the baseline of 1-3%. Our data adds to previous research showing that macrolide antibiotics, as a group, are generally safe in pregnancy and provides an evidence-based option for health professionals caring for populations with chlamydia.

A prospective-controlled study of pregnant veterinary staff exposed to inhaled anesthetics and x-rays.

Most veterinary staff are women of reproductive age. They are exposed to "waste" anesthetic gas and ionizing radiation in their workplace, which may endanger fetal safety. Presently, exposure of female veterinary staff to these health hazards has not been adequately addressed in the medical literature. Our primary objective was to investigate the incidence of major malformations associated with occupational exposure to inhaled anesthetics and/or radiation among pregnant veterinary staff. The secondary objective was to determine the rates of other adverse outcomes. We prospectively collected data on and followed-up women occupationally exposed to inhaled anesthetics and/or radiation in veterinary practices in Ontario, and compared them to controls matched for maternal age and gestational age at the time of call to the Motherisk Program. A total of 95 women were prospectively enrolled and followed-up. Among the participants there were 87 (93.5%) and 88 (92.8%) livebirths in the study and control groups, respectively. There were 4 (4.8%) major birth defects in the study group and 3 (3.4%) in the control group. The rates of spontaneous abortion were also similar, 6 (6.4%) cases in the study group and 7 (7.4%) cases in the control group. These results suggest that Ontario female veterinary staff exposed to inhaled anesthetics and/or radiation do not seem to be at an increased risk for major malformations above baseline risk.