Possible roles of nonparenchymal cells in hepatocyte proliferation induced by lead nitrate and by tumor necrosis factor alpha

Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.     

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.     

Differential expression of p53, p21waf1/cip1 and hdm2 dependent on DNA damage in Bloom's syndrome fibroblasts

The Bloom's syndrome gene, BLM, encodes a protein which bears homology to the RecQ helicases. It is believed to be involved in DNA replication and has been implicated in the maintenance of genomic stability. To investigate whether BLM was involved in cellular responses to DNA damage Bloom's syndrome fibroblasts were treated with either UV or ionizing radiation and the levels of p53 and two of its down stream effectors, p21waf1/cip1 and hdm2, were determined by western blot analysis. Following 20 J/m2 UVC-radiation we observed that the maximal accumulation of p21waf1/cip1 and hdm2 proteins preceded that of p53 in both a normal diploid fibroblast cell strain (GM0038) and in two Bloom's syndrome cell strains. Furthermore, the Bloom's syndrome cells demonstrated a delayed and prolonged accumulation of all three proteins and a delayed recovery of the protein levels back to pre-damage levels compared with the normal cell strain. Conversely, normal and Bloom's syndrome cell response following 2.5 Gy of ionizing radiation was quite similar for p21waf1/cip1 and hdm2, but differed significantly for p53. Maximum accumulation of p53 occurred within 2 h of damage and preceded that of p21waf1/cip1 and hdm2. These results suggest that the BLM protein may play a role in the detection of certain types of DNA damage and in the cellular response to that damage.     

Transforming growth factor-beta1 induces apoptotic cell death in cultured retinal endothelial cells but not pericytes: association with decreased expression of p21waf1/cip1

Building on earlier work by Pascual-Leone (1970) and Case (1985), Olson (1989; 1993) set out a theory showing how a series of incremental changes in capacity for "holding in mind" could account, in part, for children's acquisition of a theory of mind. Following Piaget (1951) infants were said to employ schemata for maintaining relations with objects and events in the presence of those events. At about 18 months children became capable of holding in mind an object so as to free the perceptual system to perceive a second object and form a relation between the two, allowing for what Piaget called the "symbolic function" and what Olson described as predication. At around 4 years, the period examined in the present study, children were said to acquire the ability to represent that predicative relation as a belief or as true or false. That was the stage at which children were said to possess a theory of mind. The present study tested the hypothesized relation between development of a theory of mind and increasing computational resources. Three-, four-, and five-year-old children's performance on a pair of theory of mind tasks was compared with that on a pair of dual processing tasks designed on the basis of Baddeley's (1986) model of working memory. The resulting correlations, as high as r = .64 between the tasks, suggest that changes in capacity to hold in mind allow the expression of, and arguably the formation of, a theory of mind.     

Reciprocal changes in p27(Kip1) and p21(Cip1) in growth inhibition mediated by blockade or overstimulation of epidermal growth factor receptors

Many human epithelial tumors express high levels of epidermal growth factor (EGF) receptors. A human-mouse chimeric version of anti-EGF receptor monoclonal antibody (mAb) C225, which blocks receptor activation and produces inhibition of cell proliferation, is currently being investigated in clinical trials. When cells bear high numbers of EGF receptors, either complete blockade of receptors with mAb 225 or full activation of receptors with EGF results in inhibition of proliferation. In the present study, we have explored the molecular mechanisms explaining how a receptor inhibitor, mAb 225, and a receptor activator, EGF, can both produce growth inhibition of A431 human squamous epithelial carcinoma cells. We reported previously that inhibition of A431 cells by EGF is associated with up-regulation of p21(Cip1). We now demonstrate that mAb 255-mediated inhibition is associated with up-regulation of p27(Kip1), which binds to and inactivates cyclin-dependent kinase-2 activity and produces cell cycle arrest in G1. Furthermore, inhibition by mAb 225 can be overcome by titrating the cultures with increasing concentrations of EGF, which is accompanied by a concurrent fall in the level of p27(Kip1). At properly titrated concentrations of mAb 225 and EGF, the inhibitory activities of both mAb 225 and EGF are counterbalanced and abolished. When EGF concentrations reach levels high enough to compete with mAb to produce near-saturating levels of receptor activation, p27(Kip1) falls below basal levels; however, the concomitant marked rise in the level of p21(Cip1) results in growth inhibition. Our data suggest that although p27(Kip1) and p21(Cip1) are induced and act independently, they play reciprocal roles in mediating inhibition of A431 cell growth by blockade of EGF receptors with mAb 225 and by activation of receptors with saturating concentrations of EGF.     

Wild-type but not mutant p53 activates the hepatocyte growth factor/scatter factor promoter

p53 transactivates the expression of a variety of genes by binding to specific DNA sequences within the promoter. We have investigated the ability of wild-type p53 and a non-DNA binding p53 mutant to activate the hepatocyte growth factor/scatter factor (HGF/SF) promoter using chloramphenicol acetyltransferase reporter constructs. We also used deletion sequences of the HGF/SF promoter to identify which regions, if any, were responsible for p53 binding. Our results show that wild-type but not mutant p53 activates the HGF/SF promoter when using -3000 and -755 bp upstream of the HGF/SF gene. This activation is lost when promoter sequences covering -365 and -239 bp are used. Analysis of the DNA sequence between -365 and -755 bp shows one putative p53 half-site with 80% homology to the consensus sequence and another half-site 3 bases downstream of this with 100% homology to the consensus sequence. In contrast to previously identified p53 binding DNA sequences, the downstream half-site is inverted. We propose that the HGF/SF promoter can be activated by wild-type p53 in vivo and that this could be as a result of a novel form of sequence-specific DNA binding.     

Early G1 growth arrest of hybridoma B cells by DMSO involves cyclin D2 inhibition and p21[CIP1] induction

Dimethylsulfoxide (DMSO) was shown to inhibit the proliferation of several B cell lines including Raji, Daudi, and SKW6-CL4 but the mechanisms involved in this growth arrest are still unclear. We show that in 7TD1 mouse hybridoma cells a DMSO-induced reversible G1 arrest involves inactivation of Rb kinases, cyclin D2/CDK4 and cyclin E/CDK2. This occurs by at least three distinct mechanisms. Inhibition of cyclin D2 neosynthesis leads to a dramatic decrease of cyclinD2/CDK4 complexes. This in turn enables the redistribution of p27[KIP1] from cyclin D2/CDK4 to cyclin E/CDK2 complexes. In addition, the simultaneous accumulation of p21[CIP1] entails increasing association with cyclin D3/CDK4 and cyclin E/CDK2. Thus, p21[CIP1] and p27[KIP1], act in concert to inhibit cyclin E/CDK2 activity which, together with CDK4 inactivation, confers a G1-phase arrest.     

Immediate increase in circulating hepatocyte growth factor/scatter factor by heparin

Circulating levels of hepatocyte growth factor (HGF)/scatter factor have been recently found to be increased in the early phase of myocardial infarction, and it has been hypothesized that HGF plays a role in angiogenesis and collateral vessel growth. Heparin has also been shown to enhance angiogenesis and to improve collateral blood flow. This study was designed to study the effect of heparin on the release of HGF. In an experimental study, heparin was given to rats intravenously and plasma was collected for measurements of HGF by enzyme-linked immunosorbent assay. A dose-dependent increase in circulating HGF was measured with peak levels occurring 10 min after injection of 300 units/kg of heparin (15.4+/-2.0 ng/ml after v 0. 17+/-0.14 ng/ml before injection,P<0.0001). In a subsequent clinical study, 12 patients received 3000 units of heparin during cardiac catheterization. Circulating HGF increased steeply within 3 min of the injection. Comparable changes in plasma concentrations were measured in samples obtained from femoral vein (8.7+/-3.5 after v 0. 33+/-0.07 before injection P<0.05) or artery (10.5+/-3.2 ng/mlv 0. 27+/-0.05 P<0.01), pulmonary artery (9.1+/-2.0 ng/mlv 0.36+/-0.06 ng/ml,P=0.07 ) or right atrium (8.5+/-1.6 ng/mlv 0.42+/-0.11,P<0.01). This study suggests that heparin-induced effects such as the promotion of angiogenesis may be at least partly due to the release of HGF.     

Expression and alteration of p53 and p21(waf1/cip1) influence the sensitivity of chemoradiation therapy for esophageal cancer

BACKGROUND/AIMS: In vitro analyses have been demonstrated that wild type p53 and p21(waf1/cip1) proteins regulate cellular proliferation and sensitivity of anticancer agents, however, the roles of p53 and p21(waf1/cip1) expression on the response to the chemoradiation therapy for human esophageal squamous cell cancer have not been investigated. METHODOLOGY: With an immunohistochemical method using specimens before and after the chemoradiation therapy, we investigate in this report the influence of the p53 and p21(waf1/cip1) expression on the chemoradiation therapy response or alteration of these protein expressions by chemoradiation therapy in thirteen esophageal squamous cell cancer patients who received our recently developed chemoradiation therapy. RESULTS: In the biopsy specimens before the chemoradiation therapy, 82% of the responders and 71% of patients with down T-classification had either a p53-/p21+ or p53+/p21+ phenotype. Eighty two percent and 46% of the cases with these two phenotypes showed complete or partial response and down T-classification, respectively. After the chemoradiation therapy, 73% of the responders and 71% of the patients with down T-classification had either a p53-/p21+ or p53+/p21+ phenotype. Eighty eight percent and 56% of the cases with these two phenotypes showed complete or partial response and down T-classification, respectively. Comparative analysis before and after the chemoradiation therapy revealed that four patients had an alteration of p53 and p21(waf1/cip1) expression in association with the therapeutic response. CONCLUSIONS: These results suggest that wild type p53 or p21(waf1/cip1) expression relates with and can be altered by the chemoradiation therapy, which could influence the therapeutic efficacy.     

Expression of p21(waf1/cip1) protein in transitional cell bladder tumours and its prognostic value

Selective antagonists to the Type 3 serotonin receptor (5HT3) in combination with corticosteroids are now considered the standard of care for the prevention of emesis from moderately to highly emetogenic chemotherapy. Here we address issues of optimal dose, schedule and route of administration of four currently available selectable 5HT3 antagonists. This paper utilizes an evidence based medicine approach to the literature regarding this class of drugs, emphasizing the results large, randomized, controlled trials to make formal recommendations concerning optimal use of this important new class of anti-emetic agents. We conclude that for each drug there is a plateau in therapeutic efficacy at a definable dose level above which further dose escalation does not improve outcome. Furthermore, a single dose is as effective as multiple doses or continuous infusion, and finally, emerging data demonstrate that the oral route is equally efficacious as the intravenous route of administration, even with highly emetogenic chemotherapy.     

A new p21waf1/cip1 isoform is an early event of cell response to oxidative stress

BACKGROUND: A population-based hemochromatosis screening program that uses serum transferrin saturation has been proposed, but few data exist on the number of U.S. adults that such a program would identify for further testing. OBJECTIVE: To determine the prevalence of an initially elevated serum transferrin saturation and the prevalence of concurrently elevated serum transferrin saturation and serum ferritin levels in the adult population of the United States. DESIGN: Nationally representative cross-sectional survey of the noninstitutionalized U.S. civilian population. PARTICIPANTS: 15 839 men and nonpregnant women 20 years of age and older who were examined in the third National Health and Nutrition Examination Survey (1988-1994). MEASUREMENTS: Single measurements of serum transferrin saturations and serum ferritin levels. Cut-off values used to define elevated serum transferrin saturation ranged from greater than 45% to greater than 62%. RESULTS: The prevalence of initially elevated serum transferrin saturation ranged from 1% to 6%. Approximately 11% to 22% of those with elevated serum transferrin saturation had concurrently elevated serum ferritin levels. The prevalence of elevated serum transferrin saturation was lower in women than in men when the same cut-off value was used to define elevated serum transferrin saturation. The prevalence of elevated serum transferrin saturation in non-Hispanic black persons and Mexican-Americans was similar to or slightly less than that in non-Hispanic white persons. The prevalence of elevated serum transferrin saturation in persons 20 to 49 years of age was as high as or higher than that in older adults. CONCLUSIONS: A hemochromatosis screening program that uses a cut-off value of greater than 60% to define elevated serum transferrin saturation would identify an estimated 1.4 to 2.5 million U.S. adults for further testing.     

Modulation of motility and proliferation of glioma cells by hepatocyte growth factor

Invasive proliferation is a critical biological characteristic of gliomas. We evaluated the activities of hepatocyte growth factor (HGF) on proliferation and motility of glioma cells, comparing them with the effects of other growth factors (EGF, bFGF, PDGF-BB, TGF-beta 1). Seven primary culture lines all expressed c-met and HGF mRNA, and secreted HGF. HGF stimulated 3H-thymidine uptake of every glioma cell line (30 to 70% upregulation). Boyden chamber assay and scattering assay revealed that HGF promoted cell motility with chemokinetic and strong chemotactic activities. Concentric circle assay showed that HGF promoted two-dimensional expansion (proliferation and motility) most strongly among the growth factors studied. Further, we analyzed 23 paraffin-embedded sections of surgically resected gliomas (7 grade II, 8 grade III, and 8 grade IV) by immunohistochemistry. Expression of HGF and Met increased with malignant progression of gliomas, suggesting that gliomas stimulated their invasive proliferation by autocrine HGF production. Neurons and vasculature were HGF-positive, and Met-positive glioma cells gathered around them. The data indicate that neurons and vasculature, which are the main tracks of glioma invasion, augment chemotactic invasion and proliferation of gliomas by paracrine HGF secretion. Clearly HGF plays a critical role in invasive proliferation of glioma cells and it is therefore a candidate target of therapeutic intervention.     

Abnormal accumulation of copper in LEC rat liver induces expression of p53 and nuclear matrix-bound p21(waf 1/cip 1)

The LEC rat is an inbred mutant strain which spontaneously develops liver injury and subsequent liver cancer. Liver injury in LEC rats has recently been shown to be closely related to abnormal copper accumulation in the liver. Previously, we reported that LEC rat hepatocytes lose their growth potential, probably allowing selective growth of preneoplastic cells. In this study, to elucidate the effects of copper accumulation on the growth activity of LEC rat hepatocytes, we examined the growth activity and the expression of p53 and p21(waf 1/cip 1) in the livers of LEC rats fed on either a control or a low-copper diet. Potential for cell proliferation of hepatocytes obtained from normal diet fed LEC rats was almost comparable to that of the cells from age-matched Sprague-Dawley (SD) rats. Northern blot analysis showed that the expression of p53 and p21(waf 1/cip 1) was significantly high in the livers of LEC rats fed a control diet, while the expression of p53 and p21(waf 1/cip 1) in the LEC rats fed a low-copper diet was as low as that of SD rat livers. Western blot analysis consistently showed that the amount of p21(waf 1/cip 1) bound to the nuclear matrix scaffold of the LEC rat liver was reduced by feeding a low-copper diet. These findings suggest that abnormal accumulation of copper induced the expression of p53 and p21(waf 1/cip 1), resulting in the inhibition of cell proliferation of LEC rat hepatocytes.     

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.