Novel NALCN biallelic truncating mutations in siblings with IHPRF1 syndrome.

Infantile hypotonia with psychomotor retardation and characteristic facies-1 (IHPRF1) is a severe autosomal recessive neurologic disorder with onset at birth or in early infancy. It is caused by mutations in the NALCN gene that encodes a voltage-independent, cation channel permeable to NM, K+ and Ca2+ and forms a channel complex with UNCSO and UNC79. So far, only 4 homozygous mutations have been found in 11 cases belonging to 4 independent consanguineous families. We studied a Sardinian family with 2 siblings presenting dysmorphic facies, hypotonia, psychomotor retardation, epilepsy, absent speech, sleep disturbance, hyperkinetic movement disorder, cachexia and chronic constipation. Polymorphic generalized seizures started at 4 and 6 years, respectively. Anti-epileptic drugs (AEDs) therapy was efficient for female proband's epilepsy, but the male still has weekly seizures. Whole exome sequencing identified 2 novel truncating mutations in NALCN allowing to assess the clinical phenotype to IHPRF1. This is the fifth family reported worldwide, and these are the first European cases with IHPRF1 syndrome with biallelic truncating mutations of NALCN.

Proteomics study of medullary thyroid carcinomas expressing RET germ-line mutations: identification of new signaling elements.

Proteomics may help to elucidate differential signaling networks underlying the effects of compounds and to identify new therapeutic targets. Using a proteomic-multiplexed analysis of the phosphotyrosine signaling together with antibody-based validation techniques, we identified several candidate molecules for RET (rearranged during transfection) tyrosine kinase receptor carrying mutations responsible for the multiple endocrine neoplasia type 2A and 2B (MEN2A and MEN2B) syndromes in two human medullary thyroid carcinoma (MTC) cell lines, TT and MZ-CRC-1, which express the RET-MEN2A and RET-MEN2B oncoproteins, respectively. Signaling elements downstream of these oncoproteins were identified after treating cells with the indolinone tyrosine kinase inhibitor RPI-1 to knock down RET phosphorylation activity. We detected 23 and 18 affinity-purified phosphotyrosine proteins in untreated TT and MZ-CRC-1 cells, respectively, most of which were shared and sensitive to RPI-1 treatment. However, our data clearly point to specific signaling features of the RET-MEN2A and RET-MEN2B oncogenic pathways. Moreover, the detection of high-level expression of minimally phosphorylated epidermal growth factor receptor (EGFR) in both TT and MZ-CRC-1 cells, together with our data on the effects of EGF stimulation on the proteomic profiles and the response to Gefitinib treatment, suggest the relevance of EGFR signaling in these cell lines, especially since analysis of 14 archival MTC specimens revealed EGFR mRNA expression in all samples. Together, our data suggest that RET/EGFR multi-target inhibitors might be beneficial for therapy of MTC.

Inactivation of Ret/Ptc1 oncoprotein and inhibition of papillary thyroid carcinoma cell proliferation by indolinone RPI-1.

Genetic alterations causing oncogenic activation of the RET gene are recognized as pathogenic events in papillary and medullary thyroid carcinomas. Inhibition of Ret oncoprotein functions could thereby represent a specific therapeutic approach. We previously described the inhibitory activity of the 2-indolinone derivative RPI-1 (formerly Cpdl) on the tyrosine kinase activity and transforming ability of the products of the RET/PTC1 oncogene exogenously expressed in murine cells. In the present study, we investigated the effects of RPI-1 in the human papillary thyroid carcinoma cell line TPC-1 spontaneously harboring the RET/PTC1 rearrangement. Treatment with RPI-1 inhibited cell proliferation and induced accumulation of cells at the G2 cell cycle phase. In treated cells, Ret/Ptc1 tyrosine phosphorylation was abolished along with its binding to Shc and phospholipase C(gamma), thereby indicating abrogation of constitutive signaling mediated by the oncoprotein. Activation of JNK2 and AKT was abolished, thus supporting the drug inhibitory efficacy on downstream pathways. In addition, cell growth inhibition was associated with a reduction in telomerase activity by nearly 85%. These findings in a cellular context relevant to the pathological function of RET oncogenes support the role of Ret oncoproteins as useful targets for therapeutic intervention, and suggest RPI-1 as a promising candidate for preclinical development in the treatment of thyroid tumors expressing RET oncogenes.

Cell cycle checkpoint efficiency and cellular response to paclitaxel in prostate cancer cells.

BACKGROUND: Defects in the cell cycle machinery of prostate cancer cells might impair the efficiency of cell cycle checkpoints and affect the cell response to chemotherapeutic drugs. We examined the relationship between the status of microtubule damage-activated checkpoints and the response of hormone-refractory prostate cancer cells to paclitaxel. METHODS: The two cell lines DU145 and PC3 harboring defects at proteins involved in the regulation of checkpoints activated by microtubule damage were examined for cell sensitivity, apoptotic response, and efficiency of checkpoints in response to paclitaxel. RESULTS: In spite of a comparable sensitivity to the antiproliferative effects of paclitaxel, DU145 and PC3 cells exhibited different cell cycle control at checkpoints activated by microtubule damage. A transient mitotic arrest was induced by the taxane in both cell lines. However, PC3 cells underwent a rapid mitotic slippage and displayed a defective postmitotic checkpoint as evidenced by the appearance of polyploid cells. In this cell line, paclitaxel-induced cell death was a slow and delayed event, occurring also after S-phase re-entry. The mitotic checkpoint appeared to be more stringent in DU145 cells compared to PC3 cells. Moreover, despite the expression of mutated proteins involved in the prevention of DNA endoreduplication (p53, pRb, and p16(INK4A)), these cells did not progress into the cell cycle but efficiently underwent apoptosis by 24 hr. Such a response of DU145 cells was associated with phosphorylation of the p21(WAF1) protein. CONCLUSIONS: These observations evidence that activation of checkpoints following microtubule damage in prostate cancer may be regulated through complex mechanisms possibly involving p21(WAF1). Our findings support that the status of cell cycle checkpoints might affect the modality of cell death. However, the relevance of the mode of cell death for the sensitivity to taxanes remains to be determined.

Clavilactones, a novel class of tyrosine kinase inhibitors of fungal origin.

Targeting of deregulated protein tyrosine kinases has been proposed as a new approach in the therapeutic intervention against pathological processes including proliferative disorders and cancer. Using a screening approach based on a comparative evaluation of antiproliferative effects in a panel of tumor cells with differential expression of protein tyrosine kinases, three benzoquinoid macrolidic fungal metabolites produced by Clitocybe clavipes, clavilactones A, B, and D (CA, CB, and CD) and two semisynthetic derivatives of these products, diacetyl-CA and dimethyl-CA, were identified as inhibitors of protein tyrosine kinases. Naturally occurring CA, CB, and CD showed inhibitory activity in kinase assays against the Ret/ptc1 and epidermal growth factor receptor (EGF-R) tyrosine kinases, while being less effective against the v-Abl tyrosine kinase and p34(cdc2) serine/threonine kinase (IC(50) 2.8, 5.5, 81.3, and 128 microM respectively, for the most potent compound CD). CB was shown to be a non-competitive inhibitor of EGF-R with respect to ATP or poly(Glu(6)Ala(3)Tyr). CD also preferentially inhibited the growth of A431 cells, which overexpress a constitutively active EGF-R, as opposed to IGROV-1 and SKOV-3 cells, which express low levels of the receptor. Further, EGF-R was shown to be a target for clavilactones in A431 cells, since EGF-induced receptor autophosphorylation was inhibited in the presence of CB, CD, and diacetyl-CA. Both CD and diacetyl-CA displayed weak activity when administered daily (i.p.) to mice bearing ascitic A431 tumor. These findings indicate that clavilactones represent the prototypes of a new structural class of tyrosine kinase inhibitors deserving further investigation.

Can deferoxamine be considered an ototoxic drug?

Some uncertainty about deferoxamine ototoxicity is to be found in the literature. Therefore, 100 patients affected by beta-thalassemia were checked audiologically. Twelve of them showed a sensorineural hearing impairment which in most cases was confined to 4 and 8 kHz. If these results are compared with a sample of "normal" population, no significant difference can be observed, therefore excluding that deferoxamine, at least at present dosages, may be considered as a certain cause of cochlear impairment.

Glucose-6-phosphate dehydrogenase deficiency and blood groups in northern Sardinia.

Glucose-6-phosphate dehydrogenase deficiency, A1A2BO, Rhesus and Kell systems were investigated in a sample of 28,439 blood donors native of northern Sardinia. The frequency of deficient male individuals was 6.56%. Higher values of r, D, and CDe compared to the averages for continental Italy were observed. Variations within the island were found in the A1A2BO and Rhesus blood group systems.

[Nutritional habits of homozygote beta-thalassemic subjects]. / Indagine sulle abitudini alimentari di soggetti beta-talassemici omozigoti.

An investigation on eating habits of a group of 40 patients with beta-thalassemia major has been carried out mainly to evidentiate iron intake whose absorption is strikingly increased in this disease. Questionnaires were used to obtain information. Daily food intake, the mean consumption of various types of food and the level of the more significant nutrients were calculated, using the National Institute of Nutrition tables. A high consumption of meat and a low consumption of fruits and vegetables were observed. A high proportion of proteins (particularly of animal origin) and a low proportion of lipids and carbohydrates was observed in comparison with the recommended levels of nutrients. Iron intake was slightly lower but its absorption may be increased because of the high proportion of calories of animal origin. A manipulation of the diet seems to be necessary to reduce the risk of iron loading.

Plasma lipids and lipoproteins pattern in beta-thalassemia major.

We studied serum lipids and lipoproteins in 20 patients with beta-thalassemia major, under high transfusion programme and regular chelation therapy, and in 20 control subjects. Total cholesterol, HDL-cholesterol, HDL2-and HDL3-cholesterol, apolipoprotein A and B levels were significantly lower in patients with Cooley's anemia, whereas free cholesterol, triglycerides and the HDL-/HDL3-cholesterol ratio did not differ in the two groups. We think that liver damage plays an important role in determining the altered lipoprotein pattern in beta-thalassemia major. However, other factors may contribute to cause such lipid changes.