Growth properties and growth factor responsiveness in skin fibroblasts from centenarians

Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.     

Induction of micronuclei in Syrian hamster embryo cells: comparison to results in the SHE cell transformation assay for National Toxicology Program test chemicals

Sixteen chemicals currently being tested in National Toxicology Program (NTP) carcinogenicity studies were evaluated in the Syrian hamster embryo (SHE) cell in vitro micronucleus assay. Results from these studies were compared to the results from the SHE cell transformation assay for the same chemicals The overall concordance between induction of micronuclei and transformation of SHE cells was 56%, which is far lower that the 93% concordance between these two tests reported previously by Fritzenschaf et al. (1993; Mutation Res. 319, 47-53). The difference between our results appears to be due to differences in the types of chemicals in the two studies. Overall, there is good agreement between the SHE cell micronucleus and transformation assays for mutagenic chemicals, but, as our study highlights, the SHE cell transformation assay has the added utility of detecting nonmutagenic carcinogens. The utility of a multi-endpoint assessment in SHE cells for carcinogen screening is discussed.     

Inhibition of EGF-dependent mitogenesis by prostaglandin E2 in Syrian hamster embryo fibroblasts

Efficient syntheses of 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine and 4-carboxy-L-phenylalanine within the context of the pentapeptide Ac-Ile-X-Gly-Glu-Phe-NH2 (wherein X = the unnatural amino acid) illustrate the use of a divergent synthetic strategy from an advanced common peptide intermediate to more readily access peptide-based tyrosine kinase inhibitors. The key intermediate, Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2, was synthesized by a facile palladium-catalyzed carbonylation of Ac-Ile-Phe(4-iodo)-Gly-Glu(O-tBu)-Phe-NH2. Oxidation of Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2 with tetrabutylammonium permanganate or addition of di-t-butylphosphite, both followed by trifluoroacetic acid deprotection, gave the target pentapeptide inhibitors wherein X = 4-carboxy-L-phenylalanine or 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine, respectively. These two peptides gave somewhat more potent inhibition of the tyrosine kinase pp60c-src than the corresponding pentapeptide wherein X = L-phenylalanine, demonstrating that appended functionalities at the 4-position are accepted and can enhance binding through added interactions within the catalytic region of the active site.     

Growth of syngeneic transplantable tumors in Syrian hamsters immunized with hamster embryonic tissue

The effect of immunization of Syrian hamsters with the hamster embryonic tissue on the growth of two syngeneic transplantable tumours was studied. Immunization of the inbred hamsters with embryonic hamster tissue with subsequent transplantation to them of transplantable syngeneic tumours led neither to inhibition nor to enhancement of the tumour growth. It is suggested that the embryonic antigens do not participate in the formation of the transplantation antitumour immunity.     

Computerized image analysis of morphologically transformed and nontransformed Syrian hamster embryo (SHE) cell colonies: application to objective SHE cell transformation assay scoring

We have developed an automated image analysis system that provides comparable classification of morphologically transformed SHE cell colonies to the current visual classification method used in the in vitro SHE cell transformation assay. Visual classification of morphologic transformation in this assay has been shown to accurately predict the carcinogenic potential of chemical, biological and physical agents. The image analysis system is quantitative, based on measuring features of colony color, texture and growth patterns. A linear combination of feature measurements produces a classification process that agrees with visual assessment 93% of the time. All identifiable sources of error are explored and the method is found to be robust in analyzing nearly 500 colonies from a variety of studies performed over a one year period. The high degree of correlation between the visual classification and the objective measurements of the image analysis system validates the reproducibility of the visual scoring process and serves as a basis for automation of the assay.     

Growth factor receptor expression during in vitro differentiation of partially purified populations containing murine stem cells

The Word Accentuation Test assesses the accentuation of 30 infrequent Spanish words written without the accentuation mark and is an easy-to-use tool for estimating premorbid intelligence of Spanish-speaking people. Its intraobserver (0.97) and interobserver (0.93) reliabilities and its correlation with the Wechsler Adult Intelligence Scale (.837) and Raven's Progressive Matrices (.655) are high, offering a good prediction of general intelligence. It is resistant to mental deterioration; 20 demented and 40 controls matched by sex, age, and education obtained similar scores. The discrepancies between current and predicted scores in Raven's scale can diagnose mild-moderate dementia with 0.79 accuracy (sensitivity, 0.78; specificity, 0.82).     

Altered [Ca2+] homeostasis in PC12 cells after nerve growth factor deprivation

[Ca2+]i homeostasis in individual PC12 cells after elevated [K+]o was studied by ratiometric microscopy, during nerve growth factor (NGF) deprivation. A significantly lower number of cells responded with an increased [Ca2+]i in the NGF deprived condition. Moreover, the responding cells were more deficient in regulating their [Ca2+]i back to control levels, after the transient peak. This suggests that differentiated neurons do not traverse the apoptotic program homogeneously with regard to their [Ca2+]i regulation and that NGF deprived PC12 cells have more difficulties to reduce their [Ca2+]i after influx of [Ca2+]o.     

The attachment and penetration of T7 DNA/phage in Syrian hamster embryonic cells

Bacteriophage T7 DNA can penetrate Syrian hamster embryonic cells after a mandatory initial pretreatment with DEAE-dextran. In 3 h an extracellular complex between T7DNA and the cell monolayer is formed which is equivalent to 105 T7 genomes per cell. During the ensuing 24-48 h of cell growth, an average of 102-103 T7 genomes are transported to the nucleus in 90% of the cells of the culture.     

Retroviral infection of Syrian hamster BHK cells depends on age and susceptibility toward sialidase

PURPOSE: To study the features of the motion perception (MP) and explore the worth of the clinical application of MP test in primary open angle glaucoma (POAG). METHODS: MPs were recorded from 41 patients (67 eyes) with POAG ang 56 normal subjects (112 eyes) by the MP software in PC compatible computer. Meanwhile, the MP ways and MP sites were analyzed and MP abnormal rate was compared to the clinical parameters and the visual function indices related to glaucoma. RESULTS: The results showed that the total abnormal rate of MP was 89.5% in POAG, and the abnormal rate was 81.6% in early stage of POAG, and MP abnormal degree had positive correlation with C/D, ocular tension and corrected loss variance (CLV), and negative correlation with visual acuity and mean sensitivity (MS), and no correlation with age and short-term fluctuation (SF) in the patients with POAG. CONCLUSION: These results suggest that the MP test provides a newly effective examination method in diagnosing the early POAG.     

Dysregulation of ornithine decarboxylase activity, apoptosis and Bcl-2 oncoprotein in Syrian hamster embryo cells stage-exposed to di(2-ethylhexyl)phthalate and tetradecanoylphorbol acetate

Perturbations of cell proliferation and death are considered as essential events in the process of carcinogenesis. Thus, two parameters, ornithine decarboxylase (ODC), an enzyme closely related to cell proliferation and transformation, and apoptotic phenomenon are profoundly modified. Using Syrian hamster embryo (SHE) cells, we have examined in the framework of two-stage carcinogenesis (initiation-promotion) the effects of a non-genotoxic [diethylhexylphthalate (DEHP)] or genotoxic [benzo[a]pyrene (BaP)] carcinogen or a non-carcinogenic compound [phthalic anhydride (AP)] on these parameters. Immunoreactive Bcl-2 and Bcl-xL proteins were also investigated following two-stage exposures. Whereas exposures to BaP, DEHP or AP alone did not provoke any modification of ODC activity, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), strongly increased it. Using two-stage exposure protocol (xenobiotics first, then replacement by TPA-promoter), the ODC activity was higher than that obtained with TPA alone. This superinduction of ODC activity was observed only with the carcinogenic compounds DEHP and BaP. Following the same exposure protocol, spontaneous cellular apoptosis was decreased. Furthermore, Bcl-2 oncoprotein was also upregulated approximately 8- and 11-fold for BaP and DEHP respectively; meanwhile Bcl-xL protein rate did not change. The non-carcinogenic compound AP slightly inhibited spontaneous SHE cell death without ODC superinduction. Exogenous polyamines, putrescine, spermidine and spermine diluted in the medium did not inhibit spontaneous apoptosis. Although inhibition of apoptosis was not specific of carcinogenic compound, both superinduction of ODC activity and inhibition of apoptosis via Bcl-2 upregulation, may cooperate during early stages of the carcinogenic process.     

Oncogenic Ras modulates epidermal growth factor responsiveness in endometrial carcinomas

OBJECTIVE: To compare cryotherapy, laser vaporization, and loop electrical excision for treatment of squamous intraepithelial lesions (SILs). METHODS: Women at least 18 years old with biopsy-proven SIL, negative pregnancy tests, negative findings on endocervical curettage, satisfactory colposcopy examinations, and congruent Papanicolaou smear and biopsy results were assigned randomly to treatment after stratification by SIL grade, endocervical gland involvement, and lesion size; they were evaluated 1, 4, 8, 12, 16, 20, and 24 months after treatment. Data were analyzed using chi2 statistics, logistic regression analysis, and the Cox proportional hazards model. RESULTS: Of 498 patients assigned, 108 were excluded (most because of inadequate follow-up), leaving 390 (139 cryotherapy, 121 laser vaporization, 130 loop excision) for analysis. All were followed 6-37 months (mean 16). There were no statistically significant differences in complications, persistence (disease present less than 6 months after treatment), or recurrence (disease present more than 6 months after treatment). Risk of persistent disease was higher among women with large lesions (risk ratio [RR], 18.9; 95% confidence interval [CI], 3.2, 110.6). Recurrence risk was higher among women aged 30 years and older (RR, 2.1; 95% CI, 1.2, 4.3), those with human papillomavirus type 16 or 18 (RR, 2.1; 95% CI, 1.1, 4.0), and those who had had prior treatment (RR, 2.1; 95% CI, 1.1, 3.9). CONCLUSION: The data support a high success rate with all three modalities. No significant difference in success rates was observed between the three treatments in our population. Additional attention and research should be directed toward the higher risk patients identified above.     

Long-term alterations in growth factor mRNA expression following seizures

Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in patients with AIDS. Nested PCR has been described as a sensitive and specific tool for detecting P. carinii DNA in clinical specimens. Little is known about the correlation of positive PCR results and clinical evidence of PCP in patients with different forms of immunosuppression. One hundred and thirty-six sputum samples, 26 tracheal-bronchial aspirate samples, 35 bronchoalveolar lavage samples, and 11 lung biopsy samples from (i) human immunodeficiency virus (HIV)-infected patients with AIDS, (ii) immunocompromised patients with leukemia or lymphoma, and (iii) immunocompetent control patients were investigated by a nested PCR amplifying DNA from the mitochondrial large subunit of P. carinii. All patients suffered from acute episodes of respiratory disease. The resulting data were correlated with clinical evidence of PCP. A high degree of association of positive P. carinii PCR results and clinical evidence of PCP in HIV-infected patients with AIDS was found. When calculated for bronchoalveolar lavage and lung biopsy samples, the positive and the negative predictive values of P. carinii PCR for PCP diagnosis in HIV-infected patients with AIDS were 1 and the specificity and the sensitivity were 100%. In contrast, in the group of patients with leukemia or lymphoma, the positive predictive value of the nested PCR for these materials was found to be as low as 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. No P. carinii DNA could be detected in specimens from immunocompetent patients. In summary, in contrast to patients with leukemia and lymphoma, nested PCR seems to be a sensitive and specific tool for PCP diagnosis in HIV-infected patients with AIDS.     

Malignant transformation of duct-like cells originating from acini in transforming growth factor transgenic mice

BACKGROUND & AIMS: In transgenic mice overexpressing transforming growth factor (TGF)-alpha in the exocrine pancreas, progressive pancreatic fibrosis and a transdifferentiation of acinar cells to duct-like cells occurs. The present study was undertaken to analyze this transdifferentiation process. METHODS: Pancreatic specimens were characterized using light microscopy and immunohistochemistry. Expression of the epidermal growth factor receptor (EGFR) and TGF-alpha was evaluated with slot blot and Western analysis. To identify other generic events, K-ras mutations were screened with an enriched polymerase chain reaction approach and p53 expression was detected with immunohistochemistry. RESULTS: Morphological examination revealed an aggregation of interlobular fibroblasts and a decrease in acinar cell height starting at day 14 after birth. In older animals, these acinar cells change to duct-like cells, which form tubular structures and express ductal markers. Evidence for dysplastic changes was found in 12 of 21 TGF-alpha transgenic mice older than 1 year. We also observed four malignant pancreatic tumors, which were multicentric and originated from dysplastic tubular complexes. They displayed a mixed cystic-papillary phenotype strongly positive for carbonic anhydrase activity. EGFR expression progressively increased in the transition from acinar to duct-like and transformed cells. Activating K-ras mutations could not be detected; however, tubular complexes and tumors displayed increased immunoreactivity for nuclear p53. CONCLUSIONS: These data suggest an involvement of the TGF-alpha/EGFR pathway in conjunction with other yet unknown events in pancreatic tumor development. Furthermore, these observations are in favor of an acinar-ductal carcinoma sequence. Thus, these transgenic animals will be useful to define genetic alterations associated with a transition from acinar cells to a neoplastic ductal phenotype.     

High concentration of glucose increases mitogenic responsiveness to heparin-binding epidermal growth factor-like growth factor in rat vascular smooth muscle cells

The effect of a high extracellular glucose concentration on the mitogenic response of rat vascular smooth muscle cells (SMCs) to heparin-binding epidermal growth factor-like growth factor (HB-EGF) was investigated. The mitogenic effect of HB-EGF was significantly greater in SMCs cultured in high glucose (25 mmol/L) than in cells cultured in low glucose (5.5 mmol/L) or at high osmolarity (5.5 mmol/L glucose plus 19.5 mmol/L mannitol). The mitogenic effect of epidermal growth factor (EGF), which shares the EGF receptor with HB-EGF, was not affected by glucose concentration. The mitogenic effect of HB-EGF was greater when incubated with heparan sulfate (HS) isolated from SMCs cultured in high glucose than with HS from cells cultured in low glucose. HS synthesized by cells in high glucose was of smaller molecular size and less sulfated than HS synthesized by cells in low glucose. The abundance of mRNA encoding HS-N-deacetylase/N-sulfotransferase (HS-NdAc/NST), a regulatory enzyme in the biosynthesis of HS, was decreased by high glucose in a protein kinase C-independent manner. These observations suggest that the enhanced mitogenic response to HB-EGF in SMCs cultured in high glucose may be attributable to changes in cell-associated HS. Downregulation of HS-NdAc/NST gene expression by high glucose may be related to the altered HS biosynthesis.     

12-O-tetradecanoylphorbol-13-acetate-induced inhibition of gap junctional communication is differentially regulated in a transformation-sensitive Syrian hamster embryo cell line compared to early passage SHE cells

The transformation-sensitive cell-line BPNi was more susceptible to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inhibition of gap junctional intercellular communication (GJIC) than early passage Syrian hamster embryo (SHE) cells, while the potency of TPA to down-regulate EGF-binding was similar in the two cell types. The kinetics of TPA-induced inhibition of GJIC suggested that different mechanisms may operate at high and low TPA concentrations. The initial inhibition after exposure to high TPA concentrations was followed by a recovery of GJIC. The recovery was much more pronounced in SHE than in BPNi cells. This effect could not be explained by differences in down-regulation of protein kinase C. Removal of high TPA concentrations also resulted in a faster recovery of GJIC in SHE than in BPNi cells. In addition, although forskolin induced a similar protection against the inhibitory effect of TPA on GJIC, forskolin restored GJIC blocked by TPA much faster in SHE than in BPNi cells. Thus, BPNi cells are more sensitive to TPA induced inhibition of GJIC than SHE cells, and have reduced capability to recover from down-regulated GJIC as compared to SHE cells.     

Spontaneous and asbestos-induced transformation of mesothelial cells in vitro

The processes of spontaneous and asbestos-induced transformation of rat mesothelium were studied using cell cultures obtained in the laboratory. The same changes in cell properties were established in both spontaneous and asbestos-induced transformation: change in epidermal growth factor (EGF) response, in some cases appearance of fibroblast-like cells instead of polygonal ones, appearance of multilayer cell growth foci, and ability to grow in semisolid agar. The response to fibroblast growth factor, insulin-like growth factor 1, and insulin did not change during transformation as well as the P450 system activity measured by benz(a)pyrene (BP) and 7,12-dimethylbenzanthracene (DMBA) cytotoxicity. The asbestos-induced transformation began earlier than the spontaneous one. EGF began to stimulate mesothelium proliferation instead of its inhibition at 6-7 passages in the case of asbestos-induced transformation, whereas during spontaneous transformation this change began at 9-10 passages. Elongated rather than polygonal cells appeared at 10-11 instead of 17-18 passages (this morphological change did not take place at all lines studied). The ability to grow in semisolid agar was found at 14-16 passages with asbestos and at 22-24 passages without it. The results allow us to propose the necessity of a positive EGF response for mesothelial cell transformation and the similarity of mechanisms of spontaneous and asbestos-induced transformation.     

Specific short transmembrane sequences can inhibit transformation by the mutant neu growth factor receptor in vitro and in vivo

The neu oncogene is activated by a point mutation within its transmembrane domain that results in the substitution of glutamic acid for valine at position 664, and is associated with constitutive activation of the tyrosine kinase. It has been proposed that the mutation allows for stabilization of homodimers of the receptor that are necessary for transduction of the mitogenic signal. To investigate the role of the alpha-helical transmembrane sequence in the function of neu, we constructed an expression vector to produce a variety of short transmembrane neu proteins, lacking ligand binding or intracellular kinase domains. Such sequences should interact with full-length receptors and prevent receptor dimerization and thus act as specific inhibitors of function. These small proteins all included a pentapeptide from position 661-665, which has been proposed to be necessary for packing. We show that the short transmembrane molecules are expressed at the cell surface and can retard the growth of neu-transformed cells in monolayers, as colonies in soft agar and as tumours in animals. As predicted by molecular modelling, the magnitude of inhibition depended on the nature of the packing surface, suggesting that the neu transmembrane domain is directly involved in neu protein dimerization.