Use of replication-deficient adenoviruses to study signal transduction pathways. Muscarinic responses in HSG and HT29 epithelial cell lines are mediated by G protein βγ-subunits

 HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca²⁺ ([Ca²⁺]_i) by activating phospholipase Cβ. In the present study, we have used the measurement of [Ca²⁺]_i with Fura-2 to show that these receptors are of the M₃ sub-type and that the increase in [Ca²⁺]_i triggered when they are activated is not sensitive to pertussis toxin. We have also used replication-deficient adenoviruses expressing wild-type and dominant-negative mutants of the α-subunits of the heterotrimeric G proteins, G_q and G_i2, to investigate the mechanisms by which these receptors control phospholipase Cβ. We find that the Ca²⁺ response to 100 μmol/l carbachol is not affected by increased expression of the wild-type α-subunit of G_q, but is blocked by the dominant-negative mutant of G_q and by both the wild-type and the dominant-negative mutant α-subunits of G_i2. Expression of α-subunits of G_i2 presumably blocks the response to carbachol by scavenging free βγ-subunits. We conclude that in HSG and HT29 cells, the Ca²⁺ response to M₃ receptor activation is mediated by the βγ- rather than the α-subunits of G_q. Received: 23 September 1998 / Received after revision: 15 December 1998 / Accepted: 11 January 1999     

ZAK is required for doxorubicin,a novel ribotoxic stressor,to induce SAPK activation and apoptosis in HaCaT cells

Doxorubicin is an anthracycline drug that is one of the most effective and widely used anticancer agents for the treatment of both hematologic and solid tumors. The stress-activated protein kinases (SAPKs) are frequently activated by a number of cancer chemotherapeutics. When phosphorylated, the SAPKs initiate a cascade that leads to the production of proinflammatory cytokines. Some inhibitors of protein synthesis, known as ribotoxic stressors, coordinately activate SAPKs and lead to apoptotic cell death. We demonstrate that doxorubicin effectively inhibits protein synthesis, activates SAPKs, and causes apoptosis. Ribotoxic stressors share a common mechanism in that they require ZAK, an upstream MAP3K, to activate the pro-apoptotic and proinflammatory signaling pathways that lie downstream of SAPKs. By employing siRNA mediated knockdown of ZAK or administration of sorafenib and nilotinib, kinase inhibitors that have a high affinity for ZAK, we provide evidence that ZAK is required for doxorubicin-induced proinflammatory and apoptotic responses in HaCaT cells, a pseudo-normal keratinocyte cell line, but not in HeLa cells, a cancerous cell line. ZAK has two different isoforms, ZAK-α (91 kDa) and ZAK-β (51 kDa). HaCaT or HeLa cells treated with doxorubicin and immunoblotted for ZAK displayed a progressive decrease in the ZAK-α band and the appearance of ZAK-β bands of larger size. Abrogation of these changes after exposure of cells to sorafenib and nilotinib suggests that these alterations occur following stimulation of ZAK. We suggest that ZAK inhibitors such as sorafenib or nilotinib may be effective when combined with doxorubicin to treat cancer patients.Key words: doxorubicin, ZAK, ribotoxic stressor, SAPKs, apoptosis     

The change in capacity and service delivery at public and private hospitals in Turkey: A closer look at regional differences

Background  

Substantial regional health inequalities have been shown to exist in Turkey for major health indicators. Turkish data on hospitals deserves a closer examination with a special emphasis on the regional differences in the context of the rapid privatization of the secondary or tertiary level health services.     

Melanoma/skin cancer screening in a Mediterranean country: results of the Euromelanoma Screening Day Campaign in Greece

Background Since the year 2000 a melanoma/skin cancer screening campaign has been organized annually in Greece in the context of the Euromelanoma Screening Day Campaign. Objectives We aimed to analyse the characteristics of the screened population, to recognize relevant risk factors and to identify the cases of histologically confirmed malignant melanoma (MM) in individuals with suspicious skin lesions. Methods An analysis of the completed screening forms from the years 2000–2004 was performed with respect to relevant demographic, epidemiological and clinical data. Results A total of 9723 individuals were screened, most of whom where below the age of 50 years (71%), female (59%), and of skin phototype II and III (76%). Sunburn during childhood was reported in 47% of participants, while 5% of the screened population had a personal or family history of melanoma. On clinical examination, 14.4% had actinic keratoses, 31.2% had dysplastic nevi, while 6.4% carried a presumptive diagnosis of non‐melanoma skin cancer. In the 2003–2004 screening campaign, 19 out of the 171 clinically suspicious lesions were histologically proven to be MM, the majority of which (58%) were ‘thin’ melanomas (Breslow's thickness of ≤ 1 mm) of the superficial spreading type. Conclusions Our study suggested that, a melanoma/skin cancer screening programme in a Mediterranean country, supported by an intense publicity campaign, attracted many individuals at risk for skin cancer and detected mostly thin melanomas of the superficial spreading type.