Sensitivity to transforming growth factor beta 1-induced growth arrest is common in human squamous cell carcinoma cell lines: c-MYC down-regulation and p21waf1 induction are important early events

Transforming growth factor beta 1 (TGF-beta 1) is a potent inhibitor of keratinocyte proliferation and a potential tumor suppressor of squamous cell carcinomas (SCCs). TGF-beta 1 exerts its antiproliferative effects by inhibiting key transitions required for progression from G1 to the S phase of the cell cycle, exemplified by a rapid reduction of c-MYC and inhibition of the G1 cyclin/cyclin-dependent kinases by induction of their inhibitors p21waf1, p27kip1, and p15INK4B. A significant majority of a new series of human SCC cell lines were found to be as sensitive as primary human epidermal keratinocytes to TGF-beta 1 growth inhibition. Only a minority of cell lines derived from late-stage tumors were resistant. An early and rapid increase in p21waf1 and reduction in c-MYC protein levels were important concomitants for TGF-beta 1 growth inhibition; these changes occurred exclusively in each of the sensitive cell lines. Expression of p15INK4B was found to be neither necessary nor sufficient for TGF-beta 1 growth arrest in the sensitive and resistant cell lines, respectively. TGF-beta 1 induced alterations in other cell cycle regulatory molecules, cyclin-dependent kinase 4, cyclin D1, pRB, and p27Kip1, occurred late and were dispensable in some of the sensitive cell lines. Expression of exogenous mycER fusion protein in one of the sensitive cell lines did not render the cells resistant to TGF-beta 1-induced growth arrest nor prevent p21waf1 induction or down-regulation of both c-MYC and mycER proteins. However, in TGF-beta 1-resistant subclones of sensitive mycER-expressing cells, p21waf1 was not induced, whereas both c-MYC and mycER protein levels decreased following TGF-beta 1 treatment. We conclude that TGF-beta 1 activates multiple cell cycle inhibitory pathways dependent upon p21waf1 induction and c-MYC degradation and that it does not function as a tumor suppressor in the majority of SCCs.     

Growth inhibitory concentrations of EGF induce p21 (WAF1/Cip1) and alter cell cycle control in squamous carcinoma cells

Previous studies have reported inhibition of A431 squamous carcinoma cell growth by nanomolar concentrations of epidermal growth factor (EGF), a potent mitogen for cells of epithelial origin. In this study, we examined potential mechanisms through which inhibition of keratinocyte growth mediated by EGF might occur by analysing components of the cell cycle regulatory machinery in A431, HN6 and HN30 keratinocytes in the presence of growth inhibitory or growth stimulatory doses of EGF. Treatment of cells with 25 pM EGF produced an increase in [3H]thymidine incorporation in A431, HN6 and HN30 cells, with respect to control cultures. Exposure to 2.5 nM EGF reduced [3H]thymidine incorporation in A431 cells and HN6 cells to 11% and 70% of control levels, respectively, whereas HN30 cells continued to proliferate in the presence of EGF. [3H]thymidine incorporation assays carried out over 24 h revealed repression of DNA synthesis in A431 cells after 12 h exposure to 2.5 nM EGF compared to untreated cells. Flow cytometry studies demonstrated accumulation of cells in G0/G1 after addition of 2.5 nM, but not 25 pM EGF. Western blot analysis revealed elevation of p21 (WAF1/CIP1/SDI1) protein levels in A431 and HN6 cells under growth-inhibitory conditions. Stimulatory doses of EGF did not induce p21 in these cells. Northern blot hybridization demonstrated elevated levels of p21 mRNA within 4 h of exposure of A431 cells to 2.5 nM EGF, which remained elevated above basal levels at 24 h. In vitro kinase assays demonstrated temporal differences in CDK2 and CDK6 activities which were related to EGF concentration. Immunocomplex Western blotting demonstrated increased association of p21 with CDK2 and CDK6 in A431 cells treated with 2.5 nm EGF. Furthermore, temporal alterations in the association of PCNA with p21 and with CDK6 were observed. The data indicate that p21 is a likely mediator of EGF-induced growth-inhibition, probably through mechanisms involving sequestration of PCNA and inhibition of CDK activity.     

Factor analyses of alcohol and water consumption in laboratory mice.

Tested 99 mice from 9 different genotypes for 10 consecutive days in a 2-choice situation with drinking tubes containing water and dilute ethanol. Separate recordings of alcohol and water consumption were taken according to the light cycle, at 9:30 AM and 9:30 PM on each day. The 20 measures of alcohol and water consumption were factor analyzed separately by alpha analysis with varimax rotation. 2 factors with eigenvalues greater than 1 were obtained for alcohol and 3 for water consumption. Factors differed strikingly depending on the fluid involved. Water-consumption factors reflected the nocturnal-diurnal activity cycle. Alcohol-consumption factors were related to changes over days but not to the activity cycle. (French summary) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Functional characterization in vivo of mutant p53 molecules derived from squamous cell carcinomas of the head and neck

Loss of wild-type p53, either through deletion or mutation, has been demonstrated in most squamous cell carcinomas of the head and neck (HNSCC). Whether these mutant molecules contribute to tumor progression purely through loss of wild-type functions or by growth-promoting mechanisms, however, remains unclear. To begin to address these issues, we isolated a series of p53 cDNAs from HNSCC cell lines that contain missense or nonsense point mutations, insertions, or deletions. The ability of each of these molecules to transform NIH/3T3 cells to a malignant phenotype was assessed by stable transfection and expression under the control of a strong heterologous promoter. NIH/3T3 cells transfected with pLTR6p53, which harbors an H179L missense mutation, formed large tumors rapidly (in less than 4 wk) when transplanted to athymic mice, as did cells expressing pLTR13p53, which had undergone a V173F missense mutation and an in-frame deletion of 48 bp between codons 208 and 223. Cells transfected with pLTR17p53, predicted from the nucleotide sequence to encode a severely truncated p53 corresponding to the N-terminal 56 amino acids, also formed tumors. Cells transfected with pLTR15p53, which was predicted to encode a less severely truncated molecule, formed much smaller tumors and at lower frequencies. NIH/3T3 cells transfected with pLTR12p53 (exon 7 splice donor mutant), pLTRwtp53 (wild-type p53), or vector alone failed to form tumors for up to 2 mo after transplantation. pLTR6p53-transfected cells exhibited a highly malignant phenotype with invasion of regional lymph nodes, mediastinal and lung metastases, invasion of the abdominal wall, and dissemination throughout the peritoneal cavity. Histological assessment of the tumors revealed intensely vascularized fibrosarcomas with numerous cellular atypia, including frequent and aberrant mitoses. Tumor explants were recultured, and northern blot analysis of cellular RNA confirmed that the expression of exogenous p53 was maintained in each case. These data indicate that different p53 mutants contribute to tumorigenesis by specific mechanisms. Furthermore, the results obtained by using the pLTR17p53 transfectants imply that some truncated molecules may overcome the effects of wild-type p53 to contribute to malignancy.     

Behavior-genetic analysis of mouse emotionality: I. Factor analysis.

Obtained pure-strain and F1 mice (N = 775) from a 6 * 6 diallel mating plan. Each S completed a battery of 12 tests of emotionality, requiring 1 mo. for completion. The subsequent 42 measures were factor analyzed by alpha factoring with varimax and promax rotations. 15 factors with eigenvalues >1.0 were found. 10 of these factors were interpreted as different facets of emotionality. Another factor was identified as weight, and 4 were underdetermined. The 10 emotionality factors were interpreted as motor discharge, acrophobia, underwater swimming, tunneling 1, audiogenic reactivity, food motivation, autonomic balance, territorial marking, activity level, and tunneling 2. Similarities with other major factor-analytic studies of emotionality are discussed. (21 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Methylphenidate, amygdalectomy, and active avoidance performance in the rat.

64 male Sprague-Dawley amygdalectomized and control rats were given 400 active avoidance training trials in a shuttle box. Controls received 0, 4, 8, or 16 mg/kg of methylphenidate throughout acquisition. Amygdalectomized Ss were given the 1st 200 trials without drug, followed by 200 trials with drug. Administration of methylphenidate produced an abrupt and large improvement in performance in the amygdalectomized Ss. One month after acquisition under the drug, retraining without drug revealed a significant retention effect for the 3 amygdaloid-drug groups relative to the nondrug-amygdaloid group. Results indicate that although amygdalectomy impairs the performance of avoidance responses, it does not prevent the learning or retention of such responses. Since methylphendiate appears to act primarily on dopaminergic mechanisms, the possible influence of amygdalectomy on such mechanisms is discussed. (41 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Electrospun gelatin–arabinoxylan ferulate composite fibers for diabetic chronic wound dressing application

Blends of arabinoxylan ferulate (AXF) and gelatin (GEL) at 1:1, 2:1 and 4:1 mass ratios were electrospun into composite fibrous mats as a wound-healing drug delivery platform. The composite fibers were characterized in terms of morphology, tensile properties, pore size, porosity and molecular composition. The composite fibers showed excellent cytocompatibility. Silver was impregnated into GEL-AXF nanofibers, and it was slowly released, resulting in bacterial growth inhibition as confirmed by the Kirby–Bauer disk-diffusion assay. This work establishes an electrospun arabinoxylan fibrous material platform with the potential to treat chronic diabetic wounds.