Influence of beta-adrenergic antagonists on cell proliferation rates in the kidney of untreated and diethylnitrosamine-treated male F344 rats.

Some nongenotoxic chemicals which cause kidney tumors have been shown to stimulate tubular cell proliferation. The aim of this study was to evaluate the effects of two beta-adrenoreceptor blocking agents, propranolol and atenolol, on cell proliferation rates in the kidneys of male F344 rats. Immunohistochemical expression of proliferating cell nuclear antigen (PCNA) and mitotic index have been examined in formalin-stored kidneys from F344 rats used in an initiation-promotion study of carcinogenesis. Cell proliferation rate was quantified in the proximal tubule epithelium. Non-initiated rats and rats initiated with a single dose of diethylnitrosamine (DEN, 200 mg/kg, i.p.) were continuously treated with propranolol (75-100 mg/kg) or atenolol (300 mg/kg) by gavage and were sacrificed after 2, 4, 8 or 21 months of experimentation. There were two control groups, one untreated (D1) and one given distilled water by gavage (D1). Control group D1 showed significantly lower cell proliferation rates than the D0 group. In non-initiated rats, propranolol had a weak enhancing effect on cell proliferation, most evident after 4 months, while atenolol had a clear enhancing effect most evident after 8 months of promoting regimen. Treatment with DENalone resulted in a significant increase in cell proliferation rate as compared to group D1. In DEN-initiated rats given propranolol, there was a borderline significant increase in cell proliferation rates, compared to rats given DEN alone, after 8 months of promoting regimen. Atenolol had no effect. Because of the differences in body weight gain and food consumption observed among the various groups, it is suggested that the state of nutrition may have obscured the effects of beta-blockers on cell proliferation rates.

Effect of tumor-promoting and anti-promoting chemicals on the viability and junctional coupling of human HeLa cells transfected with DNAs coding for various murine connexin proteins.

Gap-junctional intercellular communication is thought to be essential for maintaining cellular homeostasis and growth control. Its perturbation entails toxicological implications and it has been correlated with the in vivo tumor-promoting potential of chemicals. Little is known about the mechanism(s) responsible for the tumor promoters interference with the cellular coupling. Moreover, nongenotoxic carcinogens, as well as connexins (gap-junctional protein subunits), are known to be organ-/tissue-specific; this implies that the effect of different agents should be evaluated on their specific target, that is, connexin. To investigate the role of different connexins in regulating gap-junctional gating and to compare the properties of homotypic junctional channels, we evaluated the effects of tissue-specific tumor promoters and anti-promoters on the viability and intercellular coupling (dye-transfer) of HeLa cells stably transfected with cDNAs coding for connexin(cx)43, cx40, cx26 and cx32. The results demonstrate that the transfectants possess individual junctional permeabilities, differentially affected by the chemicals, they also show different sensitivities to the cytotoxic effect of the compounds. These findings confirm that connexin diversity may be responsible for the different gating properties of gap-junctional channels, being also suggestive for their separate functions and independent regulatory mechanisms.

Evaluation of the tumor-promoting activity of two beta-adrenoreceptor blocking agents, propranolol and atenolol, in liver of Fischer 344 rats.

The tumor-promoting activity of two beta-adrenoreceptor blocking agents, propranolol and atenolol, was tested in a two-stage protocol of hepatocarcinogenesis in male and female Fischer 344 rats. Propranolol is a lipophilic non-selective beta-blocker mainly eliminated via the liver; atenolol is a hydrophilic beta 1-selective blocking agent, mainly eliminated via the kidney. Animals were initiated with a single dose of diethylnitrosamine (DEN, 200 mg/kg, i.p.) and, after 17 days of recovery, were continuously treated with propranolol (75-100 mg/kg) or atenolol (300 mg/kg) by gavage for up to 21 months. Rats given phenobarbital (0.05% in the diet) were used as positive controls. After 2, 4 and 8 months of promotion, preneoplastic lesions were quantified by staining sections of liver for gamma-glutamyltranspeptidase (GGT). In non-initiated rats, neither propranolol nor atenolol influenced the development of spontaneous preneoplastic or neoplastic liver lesions. The results obtained in DEN-initiated rats given propranolol cannot be unequivocally interpreted. In the male, propranolol seemed to be ineffective. In the female, there was weak enhancement of DEN-induced GGT foci at 4 and 8 months and of neoplastic lesions thereafter. However, there was great interindividual variability in focus and tumor yields. Unfortunately, due to the high incidence of liver tumors in rats given DEN alone and the small number of propranolol-treated rats that survived until the end of the experiment, no definite conclusion can be drawn about the modifying potential of this beta-blocker on liver carcinogenesis. There was no evidence of liver tumor promotion in DEN-initiated rats of either sex given atenolol.

The protective role of high-density lipoprotein on oxidized-low-density-lipoprotein-induced U937/endothelial cell interactions.

The adherence of monocytes to the endothelium is an early event in atherogenesis. We have investigated this process by examining whether native and oxidized low-density and high-density lipoproteins could modulate this process. Only oxidized low-density lipoprotein caused a significant dose-dependent and time-dependent increase in U937 monocyte-like cell line binding to human endothelial cells, by a process which required de novo protein synthesis. Interestingly, E-selectin, intercellular adhesion molecule-1, vascular cell-adhesion molecule or P-selectin induction was not apparent in this system suggesting the presence of an alternative system for the interaction of endothelial cells with monocyte-like cells in response to oxidized low-density lipoprotein. High-density lipoprotein completely suppressed oxidized low-density-lipoprotein-induced adhesion of U937 cells to the endothelial monolayer, while oxidized high-density lipoprotein did not. These data suggest that the balance between native and oxidized lipoproteins may play a role in the formation of the atherosclerotic lesion by modulating monocyte endothelial interactions.

Endogenous interleukin 1 alpha must be transported to the nucleus to exert its activity in human endothelial cells.

We have previously shown that the signal peptideless cytokine interleukin 1 alpha (IL-1 alpha) may play a role as an intracellular regulator of human endothelial cell senescence (J. A. M. Maier, P. Voulalas, D. Roeder, and T. Maciag, Science 249:1570-1574, 1990). To investigate the potential intracellular function of IL-1 alpha, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-1 alpha, the precursor molecule IL-1(1-271) and the mature protein IL-1(113-271). The subcellular localization of the two different polypeptides was investigated directly or by using chimeric genes constructed by fusion of different fragments of the IL-1 alpha gene and the beta-galactosidase open reading frames. The IL-1(113-271) protein was cytoplasmic, while IL-1(1-271) was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-1 alpha nuclear targeting. Moreover, nuclear localization of IL-1 alpha correlates with impaired cell growth and expression of some IL-1 alpha-inducible genes. These results suggest that transport of endogenous IL-1(1-271) into the nucleus is required for it to modulate endothelial cell function.

Influence of the herbicide Linuron on growth rate and gap-junctional intercellular communication of cultured endothelial cells.

On the basis of in vivo toxicological long-term tests performed on rodents, the herbicide Linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] has been classified by the U.S. Environmental Protection Agency (EPA) (1988) as a class C carcinogen (possibly carcinogenic to humans). However, when Linuron was analyzed for genotoxicity, negative results were obtained. An epigenetic, tumor-promoting potential was hence suspected to be responsible for the oncogenic activity of the molecule. In the absence of in vivo data concerning the tumor-promoting activity of the herbicide and being well established that tumor promoters interfere with the cell growth rate and gap-junctional permeability, the effects of technical grade Linuron and of its trade formulation (Linuron 50) on these parameters were investigated. This is especially important in the case of the formulated preparation for a correct estimate of the health hazard to humans. Cytotoxicity and gap-junctional intercellular communication (GJIC) assays were performed on the endothelial cell line F-BAE GM 7373, an in vitro cell system known to be responsive to the biological effects of tumor promoters. A time- and dose-related cytotoxic effect was found for both Linuron and Linuron 50, the latter being the far more cytotoxic of the two. However, when tested at noncytotoxic concentrations over a period of 48 h, neither Linuron nor Linuron 50 altered the capacity of F-BAE GM 7373 cells to intercommunicate. On the basis of the results obtained, the possibility that Linuron and Linuron 50 are endowed with tumor-promoting activity is discussed. In vivo studies on tumor-promoting and genotoxic activity are in progress to complement the information available on the toxicological properties of Linuron and its trade preparation.

Senescence stimulates U937-endothelial cell interactions.

Progressive pathophysiologic modifications of endothelial cells are associated with aging. In vitro, endothelial cell senescence is accompanied by the failure to proliferate as well as by perturbations in gene expression. Here we show that (i) senescence enhances monoblastoid U937 cell adhesion to the endothelial monolayer; (ii) the enhanced interaction between senescent endothelial cells and U937 cells is mediated, at least in part, by the overexpression of ICAM-1; and (iii) LPS and interleukin 1 alpha, but not tumor necrosis factor alpha, are unable to stimulate the adhesion of U937 to senescent endothelial cells. Since monocyte adhesion to the endothelium is an early event in atherosclerosis, the altered adhesive properties observed in senescent cells could give insights into the formation of atherosclerotic lesions.

Expression and alternative splicing of fibronectin mRNA in human diploid endothelial cells during aging in vitro.

Different mRNAs for fibronectin arise from the variable processing of a single primary transcript. We used ribonuclease protection assay to investigate the changes occurring in fibronectin expression and the alternative splicing of mRNA precursor during aging in vitro of human diploid endothelial cells. Senescent endothelial cells release more protein and contain 4-5-fold more fibronectin mRNA than young cells. The pattern of alternative splicing of fibronectin mRNA, with the EDA and the CS1 segments largely included (35% and 77%, respectively) and the EDB segment undetectable, correlates well with previous studies at the protein level both in vitro and in vivo. No changes in the splicing pattern of fibronectin mRNA precursor were detected during endothelial cellular senescence. The increased expression of fibronectin in senescent cells may be a result of the activity of interleukin-1 alpha, which is overexpressed in senescent endothelial cells. It could be also important in vivo during aging and in atherosclerotic lesions.

An oligomer targeted against protein kinase C alpha prevents interleukin-1 alpha induction of cyclooxygenase expression in human endothelial cells.

We have previously demonstrated that interleukin-1 alpha (IL-1), a potent polypeptide mediator of immune and inflammatory responses, induces the expression of cyclooxygenase (cox) in human endothelial cells. The mechanism by which binding of IL-1 to its receptor stimulates gene expression remains unclear. Since phorbol 12-myristate 13-acetate, which directly binds and activates protein kinase C (PKC), induced cox expression, we examined the role of PKC as an intracellular mediator of IL-1 activity in human endothelial cells. IL-1 induced the translocation of PKC from the cytosol to the membrane. H7, a selective inhibitor of PKC, as well as an antisense oligomer blocking PKC alpha translation suppressed IL-1 induction of cox mRNA. These findings establish that PKC plays a role in the signal transduction pathway leading to the induction of cox in human endothelial cells.

Estro-progestinic replacement therapy modulates the levels of basic fibroblast growth factor (bFGF) in postmenopausal endometrium.

The levels of basic fibroblast growth factor (bFGF) in human endometrium do not vary during the ovulatory cycle but they increase significantly after menopause (M. Rusnati et al., Growth Factors, 3, 299-307, 1990). Here we report that cyclic estro-progestinic replacement therapy reduces the levels of bFGF in human postmenopausal endometrium to those measured in ovulatory cycle endometrium. Also, we have observed that the levels of bFGF in well-differentiated adenocarcinomas of the endometrium from postmenopausal patients are lower than those detected in their normal counterpart and are comparable to those measured in the normal cycling tissue. The data indicate that the stimulation of cell proliferation induced in postmenopausal endometrium by hormonal therapy or by neoplastic transformation is paralleled by a decrease of endometrial levels of bFGF. This may be due to an increase of the turnover rate of the growth factor in the proliferating versus the quiescent tissue and suggests a role of bFGF in the growth of normal and neoplastic human endometrium. Our data represent the first evidence that sex hormones modulate the levels of biologically active bFGF in vivo.

Biologically active synthetic fragments of human basic fibroblast growth factor (bFGF): identification of two Asp-Gly-Arg-containing domains involved in the mitogenic activity of bFGF in endothelial cells.

Synthetic peptides derived from the amino acid sequence of human basic fibroblast growth factor (bFGF) have been assayed for the capacity to exert bFGF agonist and antagonist activities in cultured endothelial cells. bFGF fragments A and C, which correspond to the sequences bFGF (38-61) and bFGF (82-101), induce a limited but statistically significant increase in cell number when administered to cultures of fetal bovine aortic endothelial GM 7373 cells and adult bovine aortic endothelial cells. The two peptides also exert a partial antagonist activity when GM 7373 cells are stimulated to proliferate by bFGF, but they do not affect cell proliferation induced by serum, epidermal growth factor (EGF), phorbol ester (TPA), or 1,2-diacylglycerol (diC8). Moreover, antibodies raised against peptides A and C specifically quench the mitogenic activity of bFGF. Peptides A and C contain the amino acid sequence Asp-Gly-Arg (DGR), which is the inverse of the cell adhesion signal sequence RGD recognized by integrins. DGR- and RGD-containing tetra- and heptapeptides inhibit the mitogenic activity exerted by bFGF and by the two active bFGF fragments. They do not affect cell proliferation induced by acidic FGF, EGF, serum, TPA, and diC8. However, neither peptides A and C, their corresponding antibodies, nor DGR-and RGD-containing peptides inhibit the binding of 125I-bFGF to its low and high affinity binding sites. The data suggest that amino acid residues 38-61 and 82-101, both containing a core DGR sequence, represent two "activation" domains of bFGF. Both domains are involved in the modulation of the mitogenic activity of bFGF without interacting directly with the bFGF receptor.

Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells.

Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic response in transformed fetal bovine aortic endothelial GM 7373 cells. A long-lasting interaction of bFGF with the cell is required to induce cell proliferation. bFGF-treated cells are in fact committed to proliferate only after they have entered the phase S of the cell cycle, 12-14 h after the beginning of bFGF treatment. Before that time, the mitogenic response to bFGF is abolished by 1) removal of extracellular bFGF by suramin, 2) addition of neutralizing anti-bFGF antibodies to the culture medium, 3) inhibition of PKC activity by the protein kinase inhibitor H-7, and 4) down-regulation of PKC by cotreatment with phorbol ester. Thus the requirement for a prolonged interaction of bFGF with the cell reflects the requirement for a prolonged activation of PKC. Similar conclusions can be drawn for the PKC activators 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol. The two molecules require 16 and 6 h, respectively, of activation of PKC to induce 50% of maximal cell proliferation. The requirement for a long-lasting activation of PKC appears to be a mechanism for the control of cell proliferation capable of discriminating among transient nonmitogenic stimuli and long-lasting mitogenic stimuli.

A six-amino acid deletion in basic fibroblast growth factor dissociates its mitogenic activity from its plasminogen activator-inducing capacity.

A recombinant deletion mutant of the 155-amino acid form of human basic fibroblast growth factor (bFGF), lacking amino acid residues 27-32 (Lys-Asp-Pro-Lys-Arg-Leu), was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose affinity chromatography. When maintained in the presence of an equimolar concentration of soluble heparin, the bFGF mutant (M1-bFGF) is as potent as bFGF in stimulating cell proliferation in normal and transformed fetal bovine aortic endothelial cells, in adult bovine aortic endothelial cells, and in NIH 3T3 fibroblasts. However, under the same experimental conditions, M1-bFGF is at least 100 times less efficient than bFGF in stimulating plasminogen activator (PA) production in endothelial cells, as assayed by chromogenic PA assay, SDS/PAGE zymography, and Northern blot analysis of urokinase-type PA mRNA. In the presence of heparin, M1-bFGF binds to bFGF plasma membrane receptors present on endothelial cells in a manner undistinguishable from bFGF. It also induces the same tyrosine phosphorylation pattern when added to NIH 3T3 cells. The data suggest that the PA-inducing activity of bFGF may depend upon a functional domain that differs from those involved in the mitogenic activity of the growth factor and that the binding of bFGF to its plasma membrane receptor may not be sufficient to induce urokinase-type PA production in endothelial cells.

Nuclear localization of endogenous basic fibroblast growth factor in cultured endothelial cells.

Indirect immunofluorescence using anti-human placental bFGF antibodies demonstrates the presence of bFGF-like reactivity in the cytoplasm and in the nucleus of adult bovine aortic endothelial cells and of normal and transformed fetal bovine aortic endothelial AG 7680 and GM 7372 cells. Biologically active immunoreactive Mr 18,000 bFGF can be isolated by heparin-Sepharose affinity chromatography from the extract of GM 7372 cell nuclei. Quantitation of bFGF content by biological and immunological methods indicates that 100,000 bFGF molecules per nucleus are present in GM 7372 cells, with nuclear bFGF corresponding to 25-30% of total cellular bFGF. Immunoprecipitation experiments demonstrate that the nuclear localization of newly synthesized bFGF occurs when GM 7372 cells are biosynthetically labeled both in the absence and in the presence of suramin, a molecule that inhibits the binding of bFGF to its plasma membrane receptor. Thus the data indicate that endogenous bFGF undergoes an intracellular sorting to the nucleus of the endothelial cell.

Synthesis of urokinase-type plasminogen activator and of type-1 plasminogen activator inhibitor in neuronal cultures of human fetal brain: stimulation by phorbol ester.

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.

Liver genotoxic activity of an epoxide derivative of the hepatocarcinogenic beta-blocker DL-ZAMI 1305.

A single administration of the sex-dependent hepatocarcinogenic beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (DL-ZAMI 1305) induces dose-dependent liver DNA damage, as evaluated by alkaline sucrose gradient analysis, in female but not in male Fisher 344 rats. A single administration of the direct mutagenic epoxide-derivative of DL-ZAMI 1305 3-methyl-2-nitro-1-(2,3-epoxypropoxy)-benzene induces dose-dependent DNA damage in the liver of animals of both sexes. However, also in this case, the genotoxic activity of the compound appears to be significantly higher in female than in male rats. A DNA-damaging capacity similar in the two sexes is instead exerted by DL-ZAMI 1305-unrelated direct mutagens, like N-methyl-N-nitrosourea (MNU) and methyl-methanesulfonate (MMS). The data confirm the sex-dependent susceptibility of rat liver to the genotoxic activity of DL-ZAMI 1305-related molecules, also in the absence of an absolute requirement for a metabolic activation of the compound.