Critical role of cyclin D3 in TSH-dependent growth of thyrocytes and in hyperproliferative diseases of the thyroid gland

We report that cyclin D3 is rate limiting for G1 progression in thyroid follicular cells and that its constitutive upregulation by chronic stimulation of the TSH/cAMP pathway plays a role in human and experimental hyperproliferative diseases of the thyroid gland. These conclusions are supported by in vitro and in vivo studies. In rat thyrocytes (PC Cl 3 cells), cyclin D3 expression is enhanced in response to activation of the TSH/cAMP pathway. Interference with the expression of G1 cyclins (in particular cyclin D3) by the antisense methodology strongly reduced TSH-dependent proliferation of PC Cl 3 cells, indicating that proper progression through G1 requires cyclin D3. Accordingly, PC Cl 3 cells engineered to overexpress cyclin D3 (PC-D3 cells) show enhanced growth rate and elude hormone-dependence and contact inhibition. Using an animal experimental model of thyroid stimulation, we demonstrate that cyclin D3 is a key mediator of TSH-dependent proliferation of thyroid follicular cells also in vivo. Cyclin D3 protein levels were higher in the thyrocytes from glands of propylthiouracil-treated rats compared with control animals. The increase in cyclin D3 expression occurred after the propylthiouracil-induced increase in TSH levels and preceded the burst of cell proliferation. Finally, we found that cyclin D3 protein is expressed in a fraction of human goiters but it is strongly overexpressed in most follicular adenomas.     

Lonidamine: efficacy and safety in clinical trials for the treatment of solid tumors

Lonidamine, a derivate of indazole-3-carboxylic acid, is an antineoplastic drug with a typical mechanism of action. Lonidamine has no function on cellular nucleic acids or protein synthesis, whereas it exerts a powerful inhibitory effect on oxygen consumption, aerobic glycolysis and lactate transport and accumulation of neoplastic cells. Nevertheless, its proven ability to modify the permeability of membranes is consistent with the possible increase of drug uptake, reverse of drug resistance and triggering of apoptotic pathway. Lonidamine has been experimentally shown to potentiate the cytotoxic effects of anthracyclines in human breast cancer cell lines and cisplatin activity in both platinum-sensitive and platinum-resistant human ovarian carcinoma cell lines. Since the specific mechanism of action and side effects are not overlapping with those of standard antineoplastic agents, combination of lonidamine with standard chemotherapy has been widely investigated for the treatment of solid tumors. Additionally, the enhancement of radiotherapy activity by lonidamine has been considered for palliative therapy of lesions from metastatic cancers. The encouraging results of phase II-III trials for the treatment of advanced breast, ovarian and lung cancer must be confirmed by larger studies. Specifically designed studies to address the role of lonidamine in the adjuvant setting are warranted. Lonidamine, a dechlorinate derivative of indazole-3-carboxylic acid, has proved to exert a powerful antiproliferative effect and to impair the energy metabolism of neoplastic cells. Herein we review the current experience on combining lonidamine and chemotherapy and/or radiation therapy in the treatment of solid tumors. Several studies have been published on this topic. The total number of trials reported in literature and length of follow-up are still insufficient to draw a firm conclusion. However, the available data demonstrate a significant role of lonidamine in modulating anthracycline and platinum compound activity.     

Restored T-cell activation mechanisms in human tumour-infiltrating lymphocytes from melanomas and colorectal carcinomas after exposure to interleukin-2

We investigated the effects of interleukin-2 (IL-2) exposure on T-cell signal transduction molecules and apoptosis markers in tumour-infiltrating lymphocytes (TIL) isolated from 20 melanoma and 16 colorectal carcinoma metastases and expanded in vitro for therapeutic reinfusion. Before IL-2 culture, TIL showed undetectable or very low levels of T-cell receptor (TCR) epsilon chain, p56(lck), Fas ligand (FasL) and Bax expression, while Bcl-2 values were elevated. Cancer cells were characterised by low or absent Fas and Bcl-2 and high Bax expression. Notably, they also expressed FasL. After 41-48 days of IL-2 culture, TCR epsilon chain and p56(lck) expression of TIL rose to median values of approximately 80 and 30% positive cells, respectively (P<0.001), FasL expression was detected in 45% cells from melanomas (P<0.001) and in 3% from colorectal carcinomas (P=0.09), and Bax-positive cells increased from 17.5 to 70% (P=0.005). Moreover, TCR zeta chain-positive cells were significantly increased from baseline (P=0.001), Bcl-2-positive cells dropped from 50 to 1% (P=0.007) and perforin content was high, while Fas expression was not significantly modified by IL-2 culture. In conclusion, our data suggest that the degree of immunosuppression in TIL from melanomas and colorectal carcinomas is very high, and the apoptosis markers' repertoire of cancer cells resembles that of immune-privileged tissue. Interleukin-2 culture appears to restore lymphocyte activation mechanisms, resulting in consistent FasL expression and perforin production.     

Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus

Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated beta-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immunofluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between T0 and T7 for Mad-1 and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between T0 and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-1 and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.     

ATM haplotypes and cellular response to DNA damage: association with breast cancer risk and clinical radiosensitivity

The ATM gene, mutated in the cancer-prone and radiation-sensitive syndrome ataxia-telangiectasia (AT), could predispose to breast cancer (BC) development and adverse radiotherapy responses. Sixteen ATM variants were genotyped in 254 BC cases, 70 of whom were adverse radiotherapy responders (RS-BC), and 312 control subjects and the ATM haplotypes were constructed. Constitutive ATM protein, cell survival, and the p53 response after exposure to ionizing radiation were compared in lymphoblastoid cell lines (LCLs) established from the BC cases, AT, and normal individuals. The tightly linked intronic ATM polymorphisms IVS22-77 T>C and IVS48 + 238 C>G, in the homozygote state were associated with increased BC risk [IVS22-77 CC versus TT odds ratio (OR), 1.67; 95% confidence interval (CI), 1.00-2.81], and in the heterozygote state with clinical radioprotection (IVS22-77 CT versus TT OR, 0.45; 95% CI, 0.24-0.85). Homozygote carriers of the G5557A variant were over-represented in RS-BC cases compared with non-RS-BC cases (OR, 6.76; 95% CI, 1.19-38.43). These three single nucleotide polymorphisms were associated with the three major ATM haplotypes present in >80% of the study population. BC LCLs treated with ionizing radiation exhibited an intermediate cell survival and p53 response between that of normal and AT LCLs, with the response in the RS-BC LCLs being more compromised than in the non-RS-BC LCLs. Our study suggests a general pattern of increased BC risk associated with carrying any one of the ATM variants studied, with a significant association being observed in individuals carrying variants on both ATM alleles (OR, 1.75; 95% CI, 1.09-2.81) and that ATM variants may impact on radiation sensitivity.     

Tr-kit promotes the formation of a multimolecular complex composed by Fyn, PLCgamma1 and Sam68

Tr-kit is a truncated form of the tyrosine kinase receptor c-kit expressed in the haploid phase of spermatogenesis. Upon microinjection, tr-kit triggers metaphase-to-anaphase transition in mouse eggs by the sequential activation of Fyn and PLCgamma1. Here, we show that tr-kit promotes the interaction of several tyrosine-phosphorylated proteins with the SH3 domain of PLCgamma1. Western blot analysis indicates that one of these proteins is Sam68, an RNA-binding protein that is known to interact with and be phosphorylated by Src-like kinases in mitosis. tr-kit promotes the association of Sam68 with PLCgamma1 and Fyn in a multimolecular complex, as demonstrated by co-immunoprecipitation of the phosphorylated forms of these proteins using antibodies directed to anyone of the partners of the complex. Expression of tr-kit potentiates the interaction of endogenous Sam68 also with the SH3 domain of Fyn. Furthermore, the subcellular localization of Sam68 is affected by tr-kit through activation of Fyn in live cells. Lastly, we show that interaction with the SH3 domain of Fyn triggers the release of Sam68 from bound RNA. Thus, our data suggest that tr-kit promotes the formation of a multimolecular complex composed of Fyn, PLCgamma1 and Sam68, which allows phosphorylation of PLCgamma1 by Fyn, and may modulate RNA metabolism.     

Clinical implications of varying degrees of vancomycin susceptibility in methicillin-resistant Staphylococcus aureus bacteremia

We conducted a retrospective study of the clinical aspects of bacteremia caused by methicillin-resistant Staphylococcus aureus (MRSA) with heterogeneously reduced susceptibility to vancomycin. Bloodstream MRSA isolates were screened for reduced susceptibility by using brain-heart infusion agar, including 4 mg/L vancomycin with and without 4% NaCl. Patients whose isolates exhibited growth (case-patients) were compared with those whose isolates did not (controls) for demographics, coexisting chronic conditions, hospital events, antibiotic exposures, and outcomes. Sixty-one (41%) of 149 isolates exhibited growth. Subclones from 46 (75%) of these had a higher MIC of vancomycin than did their parent isolates. No isolates met criteria for vancomycin heteroresistance. No differences in potential predictors or in outcomes were found between case-patients and controls. These data show that patients with vancomycin-susceptible MRSA bacteremia have similar baseline clinical features and outcomes whether or not their bacterial isolates exhibit growth on screening media containing vancomycin.