Induction Of Cell Division Research Articles

Inhibition of Urokinase Activity by the Antiangiogenic Factor 16K Prolactin: Activation of Plasminogen Activator Inhibitor 1 Expression

The N-terminal fragment of PRL (16K PRL) is an antiangiogenic factor that, in vitro, inhibits several components of angiogenesis including basic fibroblast growth factor (bFGF)-induced cell division, migration, and organization of capillary endothelial cells. An essential step in the regulation of angiogenesis is the activation of urokinase (urokinase type plasminogen activator, uPA), which in turn activates a cascade of proteases that play essential roles in endothelial cell migration and tissue remodeling. Treatment of bovine capillary endothelial cells (BBEC) with 16K PRL inhibited bFGF-stimulated urokinase activity in BBEC as detected by plasminogen substrate gel assay. 16K PRL did not appear to be acting via an effect on uPA expression because no change in messenger RNA levels were observed. However, protein levels of plasminogen activator inhibitor-1 (PAI-1), a specific inhibitor of urokinase, were increased by 16K PRL independent of the action of bFGF. The 16K PRL-induced increase in PAI-1 protein levels appear to be the result of increased expression of the PAI-1 gene. Increased production of PAI-1 induced by 16K PRL results in the formation of inactive PAI-1/uPA complexes, consistent with the observed decrease in uPA activity.

Permeable FGF-1 Nuclear Localization Signal Peptide Stimulates DNA Synthesis in Various Cell Types but Is Cell-Density Sensitive and Unable to Support Cell Proliferation

An earlier report indicated that a 26-amino-acid peptide (SA), comprised of the nuclear localization signal (NLS) of fibroblast growth factor-1 (FGF-1) and a membrane-permeable peptide, was able to stimulate DNA synthesis after it was taken up by NIH3T3 fibroblasts. Here, we report that SA, but not a mutant with the NLS motif destroyed, induced DNA synthesis in BALB/c3T3 murine fibroblasts, human vascular endothelial (HUVE) cells, and primary cultured hepatocytes, although the activity was weaker than that of FGF-1. The kinetics of SA-induced DNA synthesis and G1cyclin expression were similar to those elicited by FGF-1, indicating that SA induces cell cycle progression. Kinetic analysis also suggested that SA stimulates only a fraction of the DNA replication in BALB/c3T3 cells. At high cell densities, SA-induced G1cyclin expression and DNA synthesis were more strongly inhibited than those induced by FGF-1. SA did not induce cell division in HUVE and BALB/c3T3 cells and did not interfere with FGF-1-stimulated proliferation of HUVE cells. These results indicate that SA is able to partially induce cell cycle progression through a contact-inhibition sensitive signaling pathway, but it is insufficient to support cell mitosis. We also suggest that signaling by SA does not interfere with that of FGF-1.

Cell cycle regulation by plant growth regulators: involvement of auxin and cytokinin in the re-entry of Petunia protoplasts into the cell cycle.

In order to understand the mode of action of auxins and cytokinins in the induction of cell division, the effects of the two plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and N6-benzyladenine (BA) were investigated using mesophyll protoplasts of Petunia hybrida, cultivated in either complete medium or in medium deficient in cytokinin, auxin or both. Firstly we studied DNA synthesis, using 5-bromodeoxyuridine/bisbenzimide Hoechst/propidium iodide flow cytometry analyses and by the monitoring of histone H4 transcript levels. Roscovitine, a cyclin-dependent kinase (CDK) inhibitor, was found to block the cell cycle prior to entry into the S and M phases in the cultured P. hybrida protoplasts. This suggests that in Petunia cell there is a requirement for CDK activity in order to complete the G1 and G2 phases. Further experiments using roscovitine showed that neither 2,4-D nor BA were individually able to induce cell cycle progression beyond the roscovitine G1 arrest. We also monitored the phytohormonal induction of S phase by studying variations in transcript levels of the gene for mitogenactivated protein kinase (PMEK1) and transcript levels of the cell division cycle gene cdc2Pet. Only 2,4-D, and not BA, was able to stimulate PMEK1 gene transcription; thus, the more rapid S-phase induction in 2,4-D-treated protoplasts may be attributable to the activation of this transduction pathway. In contrast, both plant growth regulators were required to induce the appearance of cdc2Pet mRNA transcripts prior to S-phase engagement.

Isolation and characterization of a cytokinin up-regulated gene from tobacco mesophyll protoplasts.

We have isolated a cytokinin up-regulated cDNA clone, H13, from an early stage of cultured tobacco mesophyll protoplasts by a differential display method. The expression of this gene was specifically induced by natural and synthetic cytokinins including N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-type cytokinin, although the simultaneous presence of auxin was also required. It seems that the preceding treatment of the tobacco mesophyll protoplasts by auxin is necessary for the gene to respond to cytokinin. The addition of a cytokinin antagonist, compound 182, which suppressed the induction of cell division in tobacco mesophyll protoplasts, completely abolished the expression of this gene. Though the predicted gene product of H13 did not suggest us any sequences of defined functions, two domains of the predicted sequence had significant homology to several reported sequences in the data base. The gene product of H13 is proposed to have a role in regenerating cell wall in cultured protoplasts, since a cDNA clone E6, from cotton fiber cells, which has the most closely related structure to H13, has been isolated from cells which showed active cellulose synthesis. This supposition is supported by the evidence that in the absence of cytokinin, cell wall regeneration was significantly suppressed, resulting in failure of the induction of cell division. Thus, the gene product of H13 is supposed to have a role in regenerating cell walls and facilitating the progression of the cell cycle, resulting in the sustained cell division of tobacco mesophyll protoplasts.

The Trophic Action of IL-7 on Pro-T Cells: Inhibition of Apoptosis of Pro-T1, -T2, and -T3 Cells Correlates with Bcl-2 and Bax Levels and Is Independent of Fas and p53 Pathways

Signals from the IL-7R are essential for normal thymocyte development. We isolated thymocytes from early developmental stages and observed that suspensions of pro-T1, -T2, and -T3 cells rapidly died in culture. Addition of IL-7 promoted their survival, but did not induce cell division. Pro-T4 cells did not undergo rapid cell death, and their survival was therefore independent of IL-7. Death in the absence of IL-7 showed the hallmarks of apoptosis, including DNA fragmentation and annexin V binding; however, caspase inhibitors blocked DNA fragmentation, but did not block cell death. The trophic effect of IL-7 was partially inhibited by blocking protein synthesis. The p53 pathway was not involved in this death pathway, since pro-T cells from p53-/- mice also underwent cell death in the absence of IL-7. The Fas/Fas ligand pathway was not involved in cell death, since Fas-deficient pro-T cells died normally in the absence of IL-7, anti-Fas Abs did not protect cells from death in the absence of IL-7, and Fas expression was undetectable on cells at these stages. The IL-7 trophic affect correlated with increased intracellular levels of Bcl-2 and decreased levels of Bax, whereas no Bcl-X(L), Bcl-w, or Bad was detectable. Thus, maintaining a favorable Bcl-2/Bax ratio may account for the trophic action of IL-7.

Auxins and cytokinins control DNA endoreduplication and deduplication in single cells of tobacco

In single cells of Nicotiana tabacum L. an auxin-only signal induces elongation and DNA endoreduplication. A subsequent cytokinin+auxin signal induces cell division first as amitosis leading to DNA deduplication, then as mitosis coupled to normal cycling. During the hormone induced cell elongation the nuclei endoreduplicate, their DNA content keeping pace with the increasing cell volume. Ultimately cells can increase their nuclear DNA up to about 16C. When cell division is induced in these elongated cells, the large nuclei divide by amitosis in a seemingly random way as the two daughter nuclei do not always fit in the binary series of C-values. During the subsequent amitotic divisions the nuclear DNA content and the cell volume are further reduced. Finally, cells regain mitosis and start cycling on the 2C/4C level, forming microcalli from which normal plantlets can be regenerated.

SHORT COMMUNICATION Factors Affecting Protoplast Culture ofCucumis melo‘Green Delica’

Hypocotyls, cotyledons and etiolated half-expanded leaves ofCucumis melo‘Green Delica’ were used as explants for protoplast isolation and culture. Protoplasts isolated from cotyledons and etiolated half-expanded leaves cultured in Durand, Potrykus and Donn (DPD) medium supplemented with 0.9 μmbenzylaminopurine (BAP), 3.6 μm2,4-dichlorophenoxyacetic acid (2,4-D) and 1% sucrose, using the agarose bead culture method, were able to form cell walls and subsequently go through cell division. Pretreatment of half-expanded leaf explants in the dark for 14 d provided the best material for protoplast isolation and cell division. Approximately one third of protoplasts from etiolated half-expanded leaves formed microcolonies. For hypocotyl protoplasts, none of the treatments used were suitable to induce cell division. There was no significant difference between sucrose, glucose, and sucrose plus glucose, in culture media on the plating efficiency of leaf protoplasts ofC. melo‘Green Delica’; however, bigger colonies were formed in media supplemented with 1% sucrose. No shoot or whole plant regeneration was achieved. However, the methods reported here provide further information onC. meloprotoplast culture.

P53 mutations isolated in yeast based on loss of transcription factor activity: similarities and differences from p53 mutations detected in human tumors.

p53 is a transcriptional activator that plays a key role in the integration of signals inducing cell division arrest and programmed cell death. Moreover, p53 is a tumor suppressor gene, mutations of which are the most commonly detected mutations in diverse malignancies. In order to better understand the significance of p53 mutations to human cancer, we isolated mutant alleles of p53 that had lost transcription factor activity in yeast. These mutant alleles were evaluated for their precise changes, their activity against three different p53 responsive enhancers and their ability to act in a transdominant fashion to the wild type allele. While many of the mutations isolated in yeast resembled those found in human tumors, consistent with the importance of transcription factor activity for p53 in mammalian cells, the mutational spectrum obtained was dependent upon the p53 enhancer employed for the selection. Some mutations specifically inactivated p53 in yeast for a single enhancer element. Virtually all missense mutations tested had a dominant inhibitory effect on wild type p53 in yeast. Since some of these transdominant mutations are virtually unknown in human tumors we conclude that transdominance, per se, fails to predict which mutations occur frequently in cancer.

Induction of cell division in olfactory basal epithelium following intranasal irrigation with wheat germ agglutinin-horseradish peroxidase.

The lectin, wheatgerm agglutinin (WGA) conjugated to horseradish peroxidase (HRP), previously was shown to be transported into the central nervous system following application by intranasal irrigation. The current study investigated the hypothesis that uptake of molecules, such as the lectin-conjugate, by olfactory receptor cells would mimic internalization of other substances including odorants. This process would result in both premature death of receptor cells and increased turnover of their precursors, globose basal cells. Tetramethylbenzidine histochemical analysis showed the presence of significant amounts of the lectin-conjugate in both the receptor epithelium and olfactory bulb until at least 2 weeks postintranasal application. Neither supporting nor globose basal cells contained WGA-HRP, suggesting that uptake was primarily into olfactory receptor cells. Cell turnover, assessed by tritiated-thymidine (thymidine) autoradiography, increased both 1 and 2 weeks, but not 3 and 4 weeks, following intranasal irrigation with WGA-HRP. Most of the cells containing thymidine labelling appeared to be globose basal cells, although supporting cells also occasionally exhibited labelling. Survival of either mature or immature receptor cells in the epithelium, indicated by epithelial thickness and cell density of the septal epithelium, also declined following treatment. These data suggest that uptake of substances may result in cell loss from the olfactory epithelium and increased mitotic activity of basal cells.

Activation of glyoxalase I during the cell division cycle and its homology with auxin regulated genes

In several plant systems increase in glyoxalase I activity has been correlated with cell proliferation. Cell cycle studies of tobacco protoplasts indicate a rise in glyoxalase I activity prior to G2/M phase. Further, synthetic auxin, NAA, induced glyoxalase I activity and cell division significantly. This induction was specific in response to auxin only. Cytokinins alone do not induce cell division or increase enzyme activity. Analysis of glyoxalase I cDNA sequence from soybean shows significant homology with auxin inducible genes particularly Nt107 and limited but strong similarity with identified plant mitotic cyclins, implicating glyoxalase I in possible relationship with certain cell division regulating factors.

NO is not involved in the simvastatin induced cell division and differentiation in PC12 cells

Simvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor has been reported to inhibit cell division and induce neurite-like outgrowth in PC12 cells [Sato-Suzuki, I. and Murota, S., Neurosci. Lett., 220 (1996) 21–24]. In the present paper, we examined whether the induced nitric oxide (NO) in the simvastatin-treated PC12 cells is involved in the growth arrest and differentiation as reported in nerve growth factor (NGF) treated PC12 cells. Treatment of PC12 cells with simvastatin caused peripherin formation and enhanced NO production just like NGF-treated PC12 cells. Different from NGF, however, NO synthase inhibitors could not affect the growth arrest and differentiation in simvastatin-treated PC12 cells. In conclusion, NO had nothing to do with cell division and differentiation in simvastatin-treated PC12 cells.

Induction of cell growth and cell division in the intercalary meristem of submerged deepwater rice ( Oryza sativa L.)

In deepwater rice (Oryza sativa L.), the youngest internode can be induced to grow rapidly with submergence. This growth response is mediated by gibberellic acid (GA). It is based on both increased cell growth and increased cell division activity. In order to understand whether acceleration of the cell division cycle is a result of increased cell size, we have analyzed various growth and division parameters at the cellular and molecular levels. Flow-cytometric analysis of cells from the intercalary meristem showed that the S phase population increased within 4–6 h after submergence and the G2 phase population increased within 6–8 h, indicating activation of the cell division cycle at the G1→S phase transition. Expression of the G1 and S phase-specific cdc2Os-2 and histoneH3 genes was induced prior to S phase induction and also prior to induction of growth of meristematic cells which started with a lag phase of 4 h, indicating that transcript levels increased in response to submergence directly and not as a result of the submergence-induced accelerated growth of cells. Transcripts of the two mitotic cyclins cycOs1 and cycOs2 accumulated to significant levels in G2 phase. In parallel, activity of the histone H1 kinase which represents the G2/M phase-specific cyclin-dependent cdc2 kinase complex increased, supporting earlier findings that the activity of this kinase complex may, at least in part, be regulated at the level of cyclin expression. The molecular evidence presented therefore indicates that induction of cell division activity at G1→S occurs independently of cell growth. It also indicates that control of cell division through cell size is exerted at a level other than cdc2 gene expression.

Changes in Ultrastructure and Abscisic Acid Level, and Response to Applied Gibberellins inTaxus maireiSeeds Treated With Warm and Cold Stratification

Seeds ofTaxus maireiare known for their deep dormancy which can only be broken by a procedure involving warm stratification followed by cold stratification. Treatments with alternating temperatures of 25/15 or 23/11 °C (12 h light) for 6 months followed by 5 °C for 3 months were successful in overcoming seed dormancy. After 6 months of warm stratification, cytological changes observed included: enlargement of the embryo; a decrease in the number of lipid bodies; appearance of ER; and increases in mitochondria, plastids, dictyosomes, vacuoles and microbodies in the shoot apical meristem. Cold stratification following the warm treatment induced cell division, and one or two distinct nucleoli in the shoot apical meristem cells were observed. Both warm and cold stratification reduced endogenous ABA concentrations from the original 8888 pg per freshly harvested seed to 392 and 536 pg, respectively. Treatment with exogenous gibberellins after seeds had been warm-stratified showed that GA4and GA7were effective at promoting seed germination, but GA3was not. These results suggest that the strong seed dormancy ofT. maireicould be caused by a high ABA content and underdevelopment of the embryos in freshly shed seeds. We conclude that warm stratification with alternating temperatures increases the growth of embryos by cell expansion and enlargement and decreases ABA content, but seeds still remain ungerminated. Cold stratification may induce the response to GAs and initiate cell division resulting in release from physiological dormancy and subsequent germination ofT. maireiseeds.

Induction of cell division disturbances in chinese hamster lung cell line V79 by a complex mixture of polycyclic aromatic hydrocarbons and aza-heterocyclic compounds

A complex chemical mixture of heavy oil (Z1003), composed of polycyclic aromatic hydrocarbons (PAHs) and aza-heterocyclic compounds (AHCs), has been analysed for its ability to induce cell division disturbances on Chinese hamster lung cell line V79. In addition to the complex chemical mixture (Z1003), separated fractions of PAHs and AHCs (including four subfractions according to their polarity) were investigated. The complex mixture Z1003, PAH and AHC fractions and subfractions (excluding the weak polar subfraction) caused a strong dose-dependent mitotic arrest in cell cultures of V79 cells. The increase of metaphases was accompanied by a decrease of anaphases and telophases. In low concentrations of Z1003, PAH and AHC fractions, predominantly initial metaphases (dislocated chromosomes and/or chromosomes arranged in groups) were observed. In higher concentrations, additionally full C-metaphases and metaphases with condensed chromatin were detected. The results indicate a partial or total loss of the spindle apparatus with the result of an aneuploidy-inducing potential. According to the linkage of aneuploidy to malignant cell transformation and cancer development, a carcinogenic potency of the complex chemical mixture Z1003 and its fractions by a non-genotoxic or aneugenic mechanism should be taken into consideration.

A novel prespore-cell-inducing factor in Dictyostelium discoideum induces cell division of prespore cells.

In Dictyostelium discoideum strain V12M2, at a very low cell density (approximately 10(2) cells/cm2), most amoebae differentiate into prespore cells in a salt solution containing cAMP if an adequately diluted conditioned medium (CM) is provided (Oohata, A. A. (1995) Differentiation 59, 283-288). This finding suggests the presence of factor(s) released into the medium that are involved in inducing prespore cell differentiation. In the present study, we report the presence of two types of factors that function synergistically in prespore cell induction; one is a heat-stable and dialysable factor(s) and the other is a heat-labile and non-dialysable factor termed psi (psi) factor (prespore-inducing factor). We purified and characterized the psi factor. Its relative molecular mass was determined to be 106x10(3) Mr by SDS-PAGE and 180x10(3) Mr by gel filtration HPLC, respectively. These results indicate that psi factor exists as a dimer under native conditions. In addition to inducing prespore cell differentiation, psi factor induced cell division of prespore cells in submerged culture. Our results suggest that psi factor plays important roles not only in prespore cell differentiation but also in the progress of the cell cycle in the prespore pathway in normal development.

Molecular dissection and improvement of the nodule symbiosis in legumes

The initiation and development of nitrogen (N 2) fixing nodules in the roots of leguminous plants occurs by the induction of cell division and redifferentiation in the root cortex, followed by the formation of a meristem and progressive differentiation of specialized cells and tissues. During this process, competent rhizobia invade the root and become specialized N 2-fixing endosymbionts. The onset of the symbiosis is largely mediated by an exchange of diffusible signals, bacterial lipo-oligosaccharides being the main determinants of specificity and the initial inducers of plant responses. It is however the host which controls most facets of the nodulation process, including nodule morphology, efficiency, specificity and function. The dissection of plant mechanisms underlying signal-transduction during nodulation may be crucial to understand and then manipulate the symbiosis. Positional cloning or gene targeting offer strategies that promise the identification of crucial plant genes determining nodulation. The search for the nts — 1 gene that controls nodulation in soybean illustrates the challenges and limitations of positional cloning. It also shows how biotechnology can offer tools to help in the breeding of plant traits important to agriculture. Molecular dissection of the symbiosis will ultimately be used to improve N 2 fixation by molecular breeding and genetic engineering in legumes.

Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli.

The timing of the cell division in Escherichia coli is highly regulated, but its mechanism has not been identified. Previously we have found that the cfcA1 mutation uncouples DNA replication and cell division and elevates the frequency of cell division. We further analysed the structure and the role of the cfc genes of cfcA11, a derivative of cfcA1, and another cfc mutant, cfcB1. The cfc mutants divided prior to the ordinary stage of cell division and produced many small cells with nucleoid. However, the cells grew exponentially, and the length of a cell cycle and the initiation mass for chromosome replication were not altered by the cfc mutations. These properties resulted from a reduction of the period between the nucleoid division and the cell division in a cell cycle, and a compensatory lengthening of the period between the cell division and the initiation of the next round of DNA replication. CfcA11 has a mutation in glySa which encodes the alpha-subunit of glycyl-tRNA synthetase, and cfcB1 has an IS2 insertion in apaH which encodes Ap4A hydrolase. The cfc properties of both cfc mutants were suppressed by a multicopy plasmid carrying apaH+, and the intracellular level of Ap4A in cfcA1 was 15-fold higher, and cfcB1 was 100-fold higher than their parent. Experiments using wild-type cells showed that a high level of Ap4A caused early cell division, and that a low level of Ap4A caused delayed cell division. Ap4A is a signal for the induction of cell division. High levels of Ap4A are responsible for the initiation of cell division. The glyS mutation permits an efficient synthesis of Ap4A.

The G-protein G(i) regulates mitosis but not DNA synthesis in growth factor-activated fibroblasts: a role for the nuclear translocation of G(i).

GTP binding proteins, heterotrimeric molecules composed of alpha-, beta-, and gamma-subunits, are known to serve as transducers of information from seven-transmembrane receptors. Activation of G-proteins has been generally considered to involve subunit dissociation, with G(alpha) separating from G(betagamma). However, we have found a receptor activation of G(i) in proliferating cells that differs from these models and involves the subcellular translocation of the alpha-subunit from the cell periphery to the nucleus where G(i alpha) binds to chromatin for the duration of mitosis. This report describes the mechanism of G(i) activation in Swiss 3T3 cells in response to serum, thrombin, and epidermal growth factor, and describes a role for G(i2) in the cell cycle. Agonists were found to be unable to induce the physical dissociation of G(i2) subunits. The alpha- and beta-subunits of G(i2) could be coimmunoprecipitated with a G(i alpha) antibody from both the membrane and nuclear fractions of long-term activated cultures, showing that G(i alpha 2) and G(i beta) are induced to comigrate to the nucleus in response to growth factor receptor activation. G(i2) appears to be activated in part by a postreceptor signal that can be mimicked by protein kinase C activation; this signal may be responsible for the convergence of the signaling mechanisms of these distinct seven-transmembrane and tyrosine kinase receptors. We suggest that translocation of G(i alpha) to the nucleus induced by either thrombin or EGF may occur without subunit dissociation. Functional studies of the role of G(i) showed that pertussis toxin does not block DNA synthesis in Swiss 3T3 fibroblasts induced by serum or thrombin, but that cell proliferation is retarded to each. These results provide direct evidence for a novel mechanism of GTP binding protein activation and for an essential role of G(i) in the induction of cell division by a variety of growth factor receptors. G(i) can carry out this role in control of cellular proliferation through its translocation to the nucleus of mitotic cells.

Developmental changes in the gibberellin-induced growth response in stem segments of light-grown pea genotypes

The effects of GA on stem elongation were studied using segments from one tall and three dwarf light-grown pea genotypes varying in endogenous hormone content. Stem segments were cut at two distinct ages: when the fourth internode was at about 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Light microscopy and flow cytometry were used to demonstrate that GA does not induce cell division in excised pea stem segments. The growth studied here was strictly elongation. Measurement of final segment length after 48 hours and high resolution measurement of growth kinetics over 20 hours using an angular position transducer were done on segments treated with hormone solutions. Our data indicate that the action of GA on stem elongation can be classified into two distinct modes. The first, apparent in early-expansion stem segments, shows distinct growth kinetics and is independent of the endogenous IAA concentration of the segments. Quantitation of IAA by GC/MS in early-expansion segments of wild type pea incubated with gibberellin shows that an increase in IAA concentration is part of the GA response in such segments. The second mode of GA action is evinced in mid-expansion segments. Whereas there is no short term (<20 h) response to GA alone (as determined by growth kinetics), there is a long term (48 h) response whose magnitude decreases across the genotypes with decreasing endogenous hormone content. Growth responses indicate that in mid-expansion segments exogenous GA acts by enhancing IAA action but appears to be unable to augment endogenous IAA content. Contradictory reports of the response of excised stem segments to GA can be reconciled when tissue genotype and developmental stage are considered.

PDGF-AA, a Potent Mitogen for Cardiac Fibroblasts from Adult Rats

The heart responds to increased haemodynamic load with growth of the ventricles. The rise in ventricle mass is due to increasing mass of the myocytes and proliferation of fibroblasts and smooth muscle cells. The accompanying adaptation and remodelling of the interstitium, e.g. production and composition of the extracellular matrix proteins, determine a physiological or pathophysiological hypertrophy. Fibroblasts play a critical role in this process as the producers of extracellular matrix proteins. So far the growth factors involved are not well defined, and therefore we investigated the effect of platelet-derived growth factor (PDGF) isoforms on cellular proliferation of fibroblasts from adult rat hearts. Unlike other cell types of the cardiovascular system (e.g. smooth muscle cells), PDGF-AA has an extraordinarily high stimulatory effect on cell growth of these fibroblasts. It induces cell division to nearly the same extent and with the same kinetics as PDGF-BB as shown by cell number and flow cytometry. Cardiac fibroblasts do not express an unusually high number of PDGFα-receptors, (15 300 PDGFα-receptors, 24 800 PDGFβ-receptors per cell) which could explain this effect. Theα-receptors display a lower and shorter autophosphorylation after stimulation with PDGF in comparison to theβ-receptors. The activation of the MAP kinase pathway is not different after stimulation with both PDGF isoforms. Interestingly, quiescent cardiac fibroblasts contain a preactivated p70S6-kinase. The specific drug rapamycin not only inhibits the p70S6-kinase activation but also PDGF induced cell proliferation for more than 50%. Because the p70S6-kinase activation is implicated in growth regulation in this cell system, the preactivation of this kinase is discussed to be a possible explanation for the enhanced growth effect of PDGF-AA.