CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth.

BACKGROUND: Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. METHODS: To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten-/-/Cxcr2-/- and NRasQ61R/INK4a-/-/Cxcr2-/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten-/- and NRasQ61R/INK4a-/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). RESULTS: Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models. CONCLUSIONS: Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth.

Background: Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. Methods: To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven Braf V600E /Pten -/- /Cxcr2 -/- and NRas Q61R /INK4a -/- /Cxcr2 -/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in Braf V600E /Pten -/- and NRas Q61R /INK4a -/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). Results: Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1 , a key tumor suppressive transcription factor, was the only gene significantly induced with a log 2 fold-change greater than 2 in these three different melanoma models. Conclusions: Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways.

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg(-/-) mouse ES cells and human cancer cell lines. Parg(-/-) mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg(-/-) mouse ES cells. Parg(-/-) ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.

Poly(ADP-ribose) Glycohydrolase deficiency sensitizes mouse ES cells to DNA damaging agents.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg(+/-) and Parg(-/-)) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg(-/-) embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg(+/+) ES cells. Parg(-/-) ES cells exhibited the same growth rate as Parg(+/+) ES cells in culture. Sensitivity of Parg(-/-) ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and gamma-irradiation was examined by clonogenic survival assay. Parg(-/-) ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and gamma-irradiation compared with wild-type (Parg(+/+)) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo- and radiation therapies.

Hypovitaminosis D and valvular calcification in patients with dilated cardiomyopathy.

BACKGROUND: In patients with dilated (idiopathic) cardiomyopathy (DCM), little is known about the presence of valvular calcification and its association with hypovitaminosis D, which may predispose affected tissues to calcification. Our objectives were 2-fold: to conduct a retrospective assessment of echocardiographic evidence of valvular calcification in patients with DCM who were known to have hypovitaminosis D (25(OH)D <30 ng/mL) and to conduct a prospective assessment of serum 25(OH)D in patients with DCM, who had demonstrated echocardiographic evidence of valvular calcification. METHODS: The retrospective study consisted of 48 African American patients (34 men, 14 women; 52.3 +/- 1.5 years) having DCM and ejection fraction <35% with serum creatinine <2.0 mg/dL and 25(OH)D <30 ng/mL; and 20 white patients in the prospective study (20 men; 71.0 +/- 3.0 years) having DCM and ejection fraction <35% with serum creatinine <2.0 mg/dL and echocardiographic evidence of valvular calcification. In the retrospective study, a transthoracic echocardiogram was obtained to address mitral valvular and annular calcification, aortic valvular calcification, and sinotubular calcification; whereas in the prospective study, serum 25(OH)D level was monitored in patients with known valvular calcification. Serum parathyroid hormone (PTH) was monitored in both studies. RESULTS: In the retrospective study, hypovitaminosis D was found in 19 patients (31%) with valvular calcification and in whom serum PTH was increased (83 +/- 8 pg/mL). In the prospective study, 15 of 20 elderly patients (80%) with known DCM and valvular calcification were found to have hypovitaminosis D (25(OH)D <30 ng/mL), whereas serum PTH was normal (43 +/- 4 pg/mL). CONCLUSIONS: In patients with DCM without marked renal dysfunction, valvular calcification was seen more frequently and associated with hypovitaminosis D, whereas in elderly patients with valvular calcification, hypovitaminosis D is common, suggesting that the duration of vitamin D deficiency may determine the extent of valvular calcification. The role of hypovitaminosis D in the appearance of valvular calcification deserves further study.

Knowledge, attitudes and practices (KAP) among agricultural extension workers concerning the reduction of the adverse impact of pesticides in agricultural areas in Tanzania.

BACKGROUND: Extension workers are educated locally to enable them to train farmers in using improved local farming methods and imported technology, such as pesticides, to increase crop production. However, the extension workers are not adequately trained in health aspects of pesticides and hence are unable to provide adequate services to farmers with regard to safe use of pesticides. OBJECTIVE: The present study assessed the knowledge, attitudes and practices of agricultural extension workers with respect to health effects of pesticides in order to develop strategies for the control of pesticide exposure and prevention of pesticide poisoning in Tanzania. METHODS: A survey of Tanzanian agricultural extensionists was undertaken in 1991-1994 using a standardized questionnaire in face-to-face interviews. Extensionists who were district agricultural officers, assistant agricultural officers, agricultural assistants, and field assistants, working in the coffee and cotton growing areas (N = 61) within a radius of 15 km of farm, estate or village under farm worker study (reported elsewhere) were included in the survey. RESULTS: The majority of extensionists knew that pesticides could enter the human body but only a quarter perceived pesticides as a major problem in the community they served. The majority showed awareness of potential health hazards of the different pesticides used in their service areas, but they did not recognize what pesticides were responsible for poisoning. A high proportion was familiar with first aid procedures in case of pesticide poisoning; however, many procedures described were not appropriate for pesticide poisoning. CONCLUSIONS: The survey indicated that training of agricultural workers in health aspects of pesticide exposure is an important task for adequate provision of service to farmers with regard to safe use of pesticides in Tanzania. Nevertheless, the appropriate role of extensionists in reducing the health impact of pesticides would be to advocate pesticides of low toxicity or non-chemical pest control.

Acute health effects of organophosphorus pesticides on Tanzanian small-scale coffee growers.

Acute health effects of organophosphorus (OP) pesticides on coffee farmworkers in 1991-1992 in Tanzania are reported to provide a basis for concern over farmworkers being overexposed during application. Workers exposed to OP pesticides (N=133) were drawn from a population of about 240,000 coffee farmers. They were interviewed on symptoms and personal protection, and their erythrocyte acetylcholinesterase (AChE) activity was determined during both spraying and nonspraying period. AChE activities during spraying and nonspraying period were comparable (mean 32.0, SD 7.8 vs. 33.0, SD 8.7 U/g HgB, P=0.26). The prevalence of cough, headache, abdominal pain, excessive sweating, nausea, excessive salivation, diarrhea, and vomiting did not differ significantly between spraying and nonspraying periods. There was no suggestion of decreased AChE in exposed subjects who complained of OP-related symptoms compared to symptomless exposed subjects. Use of gloves, long boots, head cover, face cover, and coverall was not significantly associated with AChE activity. No marked AChE depression was found during spraying season, which may explain the lack of association between symptoms and AChE. The fact that only moderately toxic OP pesticides were used may indicate that toxicity was not sufficiently high to cause depression. Experience, however, suggests that occupational poisoning remains a potential serious danger in coffee cultivation in Tanzania.

Assessment of the ability of health care providers to treat and prevent adverse health effects of pesticides in agricultural areas of Tanzania.

A survey of Tanzanian health care providers in agricultural areas was undertaken in 1991-1994 to assess their knowledge of toxic effects of pesticides, experiences and practices, as well as of their needs for appropriate information in order to develop effective strategies for reducing pesticide poisoning. Face-to-face interviews were conducted with 104 physicians, clinical officers and nurses at health care facilities in the coffee and cotton growing areas. Eighty percent of respondents reported to have seen one and nine of them two to four cases of pesticide poisoning in the preceding three months. A significantly higher annual number of poisonings were observed in coffee than in cotton area (GM 0.5 vs 0.1). Also the number of cases registered in hospitals was considerably higher than that in the out-patient health care (GM 1.7 vs 0.2). Pesticide poisoning was regarded as a major problem in the community by 63% of health care providers, including 77% of hospital staff. One third of health care providers thought that a certain percent of pesticide poisoning cases remain unrecognized, and that this percentage is higher in cotton than in coffee growing areas. The respiratory tract was the major route for pesticide to enter the human body; this was followed by gastrointestinal tract, skin, and eyes. Only one percent of the respondents could identify the groups of pesticides (organophosphate vs organochlorine) mostly used in the study areas. The survey indicated that training of hospital staff in toxicity of pesticide exposure is an important task and a prerequisite for efficient recognition, diagnosis and treatment of pesticide poisoning cases in Tanzania.

Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins.

The measurement of nitric oxide (NO) is important for direct examination of the regulatory roles of NO in various biological systems. Diaminofluoresceins (DAFs), new fluorescence indicators for NO, were applied to detect the release of NO from bovine aortic endothelial cells (ECs). DAFs react with NO to yield the corresponding green-fluorescent triazolofluoresceins, which provide the advantages of specificity, sensitivity and a simple protocol for the direct detection of NO. Using these DAFs, we could detect the generation of NO not only from inducible NO synthase expressed in macrophages, but also from constitutive NO synthase expressed in ECs.

Sarcoid-like reaction in the regional lymph nodes and spleen in gastric carcinoma: a clinicopathologic study of five cases.

To clarify the occurrence of sarcoid-like reaction in the spleen of the gastric carcinoma patients, 100 consecutive specimens from gastrosplenectomy were examined. Sarcoid-like reaction was observed in the lymph nodes of 13 cases (13%) and the spleen of five cases (5%). All cases of the latter group were included in the former one. None of them showed any symptoms or signs indicative of systemic sarcoidosis. It seems that the cases with sarcoid-like reaction in the spleen ocurred more frequently in an advanced stage of the gastric cancer than those without this phenomenon. Epithelioid cell granulomas (EPGs) appeared to arise in the periarteriolar lymphoid sheaths of the spleen histologically, but were never found in red pulp or germinal centers. They were composed of groups of epithelioid cells and accompanied by the small lymphocytes and plasma cells. In three cases, scattered eosinophils were also observed among the epithelioid cells. Immunohistochemically, the majority of the intragranulomatous small lymphocytes had T-cell phenotype, while B-cells formed only the minor cellular population. None of the 13 cases contained EPGs in the primary tumor. Our study indicates that sarcoid-like reaction in the spleen is possibly not such a rare phenomenon in the gastric cancer as previously considered and more frequently seen in the advanced stage of the gastric cancer. Sarcoid-like reactions of the regional lymph nodes were more frequently seen in the patients with EPGs in the spleen than in those without. We also suggest that the incidence of sarcoid-like reactions in the spleen is closely related to those in pancreaticosplenic nodes and/or nodes of the hilus of the spleen.

Aggressive surgery for pancreatic duct cell cancer: feasibility, validity, limitations.

Pancreatic duct cell cancer is characterized by a low resectability rate and a low long-term survival rate. Between September 1974 and December 1992 in our institution, 149 (59%) of the 253 patients with this cancer underwent resection. The operative procedures were pancreatoduodenectomy in 105 patients, total pancreatectomy in 36, and distal pancreatectomy in 8. The tumor was extirpated with extensive dissection of the lymph nodes and excision of the nerve plexus in the retroperitoneum. Of the 149 patients, 79 (53%) underwent combined resection of the pancreas and the portal vein; 16 of the 79 patients also underwent resection of the adjacent arteries. Three patients died within 30 days after surgery, and 17 other patients succumbed within 2 to 7 months. The mortality among patients undergoing pancreatectomy and resection of the portal vein (9.5%) was similar to that of patients with pancreatectomy alone (10%). Curative resection was necessary for long-term survival. The 5-year survival rate in 61 patients with the curative resection was 15%. Ten patients lived more than 5 years. Even patients with lymph node metastases and cancer invasion of the portal vein had a prolonged survival. Intraoperative irradiation was carried out in 35 patients to improve the survival rate, but without success. Infusion chemotherapy with 5-fluorouracil via the portal vein was tried in 25 patients, resulting in a decrease in liver metastasis. We have made some progress in the first step toward improving treatment, although we are far from the goal and it is necessary to conduct additional trials.

Social intergration and change in activities of daily living in Japanese elderly.

The prospective association of social integration reported in 1987 with change in activities of daily living (ADL) over three years was examined in a cohort of 2200 Japanese elderly, aged 60 years and over. Social integration data was obtained from a national survey which measured both the quantity and frequency of attachment. Social ties were classified into five types: spouse, child(ren) living together, child(ren) living separately, friend, neighbor, and community group. Eight potential confounding variables--age, educational attainment, existence of chronic diseases, subjective well-being, drinking, sleeping hours, cigarette smoking, and exercise--were controlled using logistic regression analysis for each sex. Men who attended community group meetings more frequently had significantly less decline in ADL during the follow-up period for those who were ADL unimpaired at the baseline survey, and were also significantly more improved for those who were ADL impaired people at the baseline survey. Trends for women were not significant.

Novel lowly immunosuppressive antitumor fluorouridine derivative, UK-21: antitumor activity and effect on humoral immune response in mice.

Our previous studies indicated that a newly synthesized 5-fluorouridine derivative, 2',3',5'-tris-O-[N-(2-n-propyl-n-pentanoyl)glycyl]-5-fluorouridine (UK-21), revealed its antitumor activity by being converted to 5-fluorouridine (5-FUR) and showed a low level of immunological side effects. However, the bioavailability of UK-21 given orally did not seem to be good. In the present study, we focused on the antitumor and immunosuppressive activities of UK-21 given i.p. to mice. UK-21 suppressed the growth of L-1210, P388 and EL4 leukemias inoculated i.v. into corresponding syngeneic mice and both the growth of Lewis lung carcinoma transplanted s.c. and its subsequent metastasis to the lung. UK-21 showed antitumor activity at doses almost 10 times lower than those of 5-fluorouracil (5-FU). The side effects of UK-21, especially on immune functions, were examined in comparison with those of 5-FUR, 5-FU, and cyclophosphamide (CY) at doses producing comparable antitumor activity. The suppressive effect of UK-21 on IgM and IgG antibody formation in mice immunized with ovalbumin was clearly weaker than that of 5-FUR, 5-FU, and CY. The suppressive effect of UK-21 on thymus weight was markedly weaker than that of 5-FU and CY. The reduction of WBC counts induced by UK-21 was also lower than that produced by any other agent. The results reported herein suggest the strong possibility of UK-21 being developed as a novel anticancer drug with cytotoxic mechanisms different from those of 5-FU. Our study also points to the chemical modification of 5-FUR as a feasible way of developing new anticancer drugs.

[A study of attitudes toward medical care and health behaviors among older adults].

In order to understand the attitudes of older adults toward medical care, we interviewed 480 persons living in Yokohama, and 180 persons living in Aikawa, Kanagawa, aged 45 to 84 years old. The following results were obtained; 1) Attitudes toward medical care can be classified into four types; self-determined medical care, self treatment attitudes, high dependence on the medical care system, and distrust of medical care. Those interviewees who had high self-determination in medical care and self treatment attitudes showed strong distrust of medical care. 2) There were two groups with trend toward low compliance to the advice of a physician for a physician diagnosed illness: the group that had strong self-determination in medical care, and the group that had high distrust of medical care. The interviewees who had a strong tendency to see a physician for potentially serious illness had high self treatment attitudes, but disease prevention behaviors was not associated with all of four types. 3) In both communities, those interviewees who were younger and with higher educational levels showed strong distrust of medical care and had more self-determination attitudes. Those interviewees who had actually experienced problems in medical treatment showed less dependence on medical care and more distrust of medical care compared to those who had not. In Yokohama, distrust of medical care appeared to be higher among those interviewees who did not have a family doctor than those who had. 4) Distrust of medical care and self-determination in medical care was significantly higher in Yokohama than in Aikawa. The differences in the distribution of educational level and family doctors were a part of the reason for area differences in attitudes of distrust of medical care.