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PubMed1995

Microvessel density, endothelial cell proliferation and tumour cell proliferation in human colorectal adenocarcinomas.

BACKGROUND: Thymidine incorporation studies performed in animal tumour models, revealed major differences in endothelial cell proliferation when tumour tissue was compared with normal tissue. The fraction of proliferating endothelial cells is reported to be increased by a factor of 30 to 40 in tumour tissue. PATIENTS AND METHODS: To make it possible to analyze the endothelial cell proliferation in human tumours, an immunohistochemical double staining technique comprising CD31, an endothelial cell marker, and Ki-67, a proliferation marker, was developed. Endothelial cell proliferation was analysed in 21 primary human colorectal adenocarcinomas and in the adjacent mucosa. RESULTS: Proliferating endothelial cells were found throughout the entire carcinoma. The mean overall endothelial cell labeling index (ECLI) was 9.9% (range, 5.4-18.0), and the labeling index of endothelial cells in areas of intense neovascularisation was even higher. Mean ECLI in the vascular hot spots was 21.0% (range, 6.8-35.0), and the mean tumour cell labeling index (TCLI) in the maximally Ki-67 immunostained areas was 78.3% (range 47.0-89.7). In 14 of 21 carcinomas, these areas were predominantly found at the luminal margin of the tumour, as were the vascular hot spots. A significant positive correlation was found between tumour vascularity, measured in the vascular hot spots, and tumour cell proliferation, measured in the maximally Ki-67 immunostained areas (p < 0.05). To analyse this relation in more detail, microvessel density (MVD), TCLI and ECLI were determined per x400 microscopic field by scanning in sequence from the luminal tumour margin to the invasive tumour base. In all tumours, the pattern of the MVD per x400 field, from the luminal margin to the tumour base, was similar to that of the TCLI and ECLI. CONCLUSIONS: These findings confirm that the fraction of cycling endothelial cells is higher in human colorectal carcinoma than in the adjacent mucosa which suggests that endothelial cells are proliferating in most of the individual capillaries in tumour tissue. Regional differences in MVD correlate with differences in tumour cell proliferation in these tumours.

Adenocarcinoma↗
PubMed2006

Inhibition of smooth muscle proliferation by urea-based alkanoic acids via peroxisome proliferator-activated receptor alpha-dependent repression of cyclin D1.

OBJECTIVE: Proliferation of smooth muscle cells is implicated in cardiovascular complications. Previously, a urea-based soluble epoxide hydrolase inhibitor was shown to attenuate smooth muscle cell proliferation. We examined the possibility that urea-based alkanoic acids activate the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) and the role of PPARalpha in smooth muscle cell proliferation. METHODS AND RESULTS: Alkanoic acids transactivated PPARalpha, induced binding of PPARalpha to its response element, and significantly induced the expression of PPARalpha-responsive genes, showing their function as PPARalpha agonists. Furthermore, the alkanoic acids attenuated platelet-derived growth factor-induced smooth muscle cell proliferation via repression of cyclin D1 expression. Using small interfering RNA to decrease endogenous PPARalpha expression, it was determined that PPARalpha was partially involved in the cyclin D1 repression. The antiproliferative effects of alkanoic acids may also be attributed to their inhibitory effects on soluble epoxide hydrolase, because epoxyeicosatrienoic acids alone inhibited smooth muscle cell proliferation. CONCLUSIONS: These results show that attenuation of smooth muscle cell proliferation by urea-based alkanoic acids is mediated, in part, by the activation of PPARalpha. These acids may be useful for designing therapeutics to treat diseases characterized by excessive smooth muscle cell proliferation.

8,11,14-Eicosatrienoic Acid↗
PubMed2005

Cell proliferation activity of proliferating bile duct after bile duct ligation in rats.

The cell proliferation activity of proliferating bile ducts produced by bile duct ligation (BDL) in rats was examined histologically, immunohistochemically, and ultrastructurally. Proliferating bile ducts, which were similar to normal bile ducts, increased with time after BDL. The cell proliferation activity of proliferating bile ducts, measured using proliferating-cell nuclear antigen and 5-bromo-2'-deoxyuridine antibodies, tended to be high at 1 and 3 days after BDL and decreased progressively at 2 to 4 weeks after BDL. On the other hand, alpha-smooth muscle actin-positive myofibroblast-like cells increased continuously after BDL. These findings indicate that there is a negative correlation between the cell proliferation activity of proliferating bile ducts and that of myofibroblast-like cells.

Animals↗
PubMed1988

Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry.

Proliferating cell nuclear antigen (PCNA)/cyclin is an intranuclear polypeptide antigen that is found in both normal and transformed proliferating cells. We have recently described two mouse monoclonal antibodies reacting with PCNA. In this report we describe the application of these antibodies to the study of proliferating human cells by indirect immunofluorescence microscopy and by flow cytometry. A fixation/permeation procedure was developed in order to obtain satisfactory binding of monoclonal PCNA-specific antibodies to proliferating cells. This method involved fixation with 1% paraformaldehyde followed by methanol treatment. For the staining of cells in suspension with the IgM type monoclonal antibodies lysolecithin was added to the paraformaldehyde solution to achieve a better permeation by the antibody molecules. This procedure gave a good ratio of specific staining relative to the background staining. It also preserved the shape and normal architecture of the cells as judged by visual microscopic observation and by light scatter measurements using a flow cytometer. Furthermore, this fixation technique permits simultaneous labeling of DNA by propidium iodide and PCNA by monoclonal antibodies. PCNA was detected in various types of normal and transformed proliferating cells by indirect immunofluorescence. Quiescent peripheral blood mononuclear cells were PCNA-negative whereas a fraction of lectin-stimulated lymphocytes became PCNA-positive. Similarly, early passages of fetal skin fibroblasts were PCNA-positive but non-proliferating senescent fibroblasts of later passages were PCNA-negative. The association of PCNA-staining by monoclonal antibodies with cell proliferation was confirmed by flow cytometry. Simultaneous labeling of PCNA and DNA showed that the PCNA signal increased during the G1 phase of the cell cycle, reached its maximum in the S-phase, and declined during the G2/M phase. Using cell sorting we demonstrated that mitotic cells had a very low PCNA signal. Thus, monoclonal PCNA-specific antibodies offer a convenient tool for the detection of human cell proliferation by immunofluorescence microscopy and by flow cytometry.

Animals↗
PubMed2001

Evidence that reduction of hepatocyte growth factor (HGF) is not required for peroxisome proliferator-induced hepatocyte proliferation.

The mechanisms underlying peroxisome proliferator-induced hepatocarcinogenesis are not understood. Because of the uncertainty of human cancer risk associated with peroxisome proliferators, delineating the mechanisms of carcinogenesis by these agents is of great interest. Alterations in liver growth factors were postulated to contribute to the carcinogenic effect of peroxisome proliferators. Administration of these compounds to rodents results in down-regulation of hepatocyte growth factor (HGF) and supplementing culture medium with HGF is reported to suppress cell proliferation of preneoplastic and neoplastic cells from WY-14,643-treated livers. Combined, these observations suggest that reduced levels of hepatic HGF contribute to the mechanisms underlying peroxisome proliferator-induced hepatocarcinogenesis. To determine if HGF can prevent the effects of peroxisome proliferators in liver, the short-term influence of WY-14,643 in two different lines of HGF transgenic mice was examined. Mice were fed either a control diet or one containing 0.1% WY-14-643 for one week. Hepatomegaly was found in both HGF transgenic mouse lines fed WY-14,643 compared with controls. Additionally, hepatic expression of typical mRNA markers of peroxisome proliferation including those encoding peroxisomal fatty acid metabolizing enzymes and cell cycle control proteins were all significantly elevated in HGF transgenic mice fed WY-14,643 compared with controls. Down-regulation of HGF was found to be dependent on PPARalpha since lower levels of HGF mRNA and protein were observed in wild-type mice fed WY-14,643 for 1 week and not in similarly treated PPARalpha-null mice. These results demonstrate that the early increase in hepatic mRNAs associated with peroxisome and cell proliferation induced by WY-14,643 treatment can not be prevented by overexpression of HGF in vivo.

Animals↗
PubMed2004

Morphology of proliferating and non-proliferating tumor cell nuclei in glioblastomas correlates with preoperative data from proton-MR-spectroscopy.

In contrast to the growing interest in proton-MR-spectroscopy (1HMRS) for preoperative examination of patients with brain tumors, there is nearly no knowledge about a correlation between data from 1HMRS and histomorphology as confirmed by quantitative morphological methods. Whether a correlation can be confirmed between data from 1HMRS and quantitative histomorphology of glioblastomas representing the most frequent type of brain tumors was investigated in the present study. Furthermore, it was of interest, whether correlations between spectroscopic data and histomorphology can be confirmed for proliferating and non-proliferating tumor cell nuclei independently. Using stringent inclusion criteria for this study, 24 patients were investigated by means of preoperative 1HMRS and by means of digital image analysis of paraffin sections from the surgical specimen. Proliferating and non-proliferating tumor cell nuclei were investigated separately in the region with the highest proliferative activity in each tumor using immunohistological staining for the proliferation marker Ki67. Main results showed highly significant correlations between total creatine and variables of nuclear size, as well as correlations between choline and variables of nuclear shape. These results were confirmed for both proliferating and non-proliferating tumor cell nuclei. A significant correlation between N-acetyl-aspartate level and topometric variables (number of neighbors per nucleus, variables describing distances between tumor cell nuclei) was confirmed for proliferating tumor cell nuclei. Discriminant analysis provided a good separation of cases with high and with low values for these spectroscopic variables based on histomorphometric data. In conclusion, the results confirm a direct correlation between data from preoperative 1HMRS and histomorphological characteristics of glioblastomas supporting the biological relevance of spectroscopic data for the examination of brain tumor patients.

Cell Nucleus↗
PubMed2000

Peroxisome proliferator-activated receptor gamma transcriptional regulation is involved in platelet-derived growth factor-induced proliferation of human hepatic stellate cells.

During liver injury, hepatic stellate cells (HSC) acquire a myofibroblast-like phenotype associated with reduction of lipid droplets, increased collagen synthesis, and proliferation. Peroxisome proliferator-activated receptor gamma (PPARgamma) regulates adipocyte differentiation and controls gene transcription in response to various activators including prostanoids and antidiabetic thiazolidinediones. We explored whether the presence of PPARgamma and its transcriptional activity were involved in control of HSC proliferation in vitro. PPARgamma ligands, 15-deoxy-triangle up(1214) prostaglandin J(2) (15d-PGJ(2)) and ciglitizone, significantly decrease platelet-derived growth factor (PDGF)-induced proliferation in activated human HSC and inhibit alpha smooth muscle actin (alpha-SMA) expression during HSC transdifferentiation. Treatment with 9-cis retinoic acid (9-cisRA) and LG268, ligands of the heterodimerization partner retinoic X receptor (RXR), had a negligible effect in PDGF-treated cells but caused a further reduction of proliferation when used in combination with ciglitizone. Transfection experiments with a reporter gene consisting of 3 copies of a PPAR response element (peroxisome proliferator response element [PPRE](3)-tk-luciferase) showed a progressive reduction of PPAR transcriptional activity during plastic-induced HSC transdifferentiation. Cotransfection with human PPARgamma expression vector restored the PPRE(3)-tk-luciferase reporter expression and the increased level of the receptor in activated HSC-inhibited cell proliferation in a dose-dependent manner. Incubation of human PPARgamma-cotransfected HSC with PDGF strongly inhibited luciferase activity and this effect was blocked by the inhibition of the mitogen-activated protein (MAP) kinase signal cascade. Our results indicate that depression of PPARgamma expression and activity is involved in HSC proliferation and that the PPARgamma ligand-mediated activation exerts a previously unrecognized inhibition of PDGF-induced mitogenesis in activated human HSC.

Cell Differentiation↗
PubMed1984

Distribution of acid hydrolases in subcellular fractions of proliferating vs non-proliferating fibroblasts.

We have employed colloidal silica (Percoll) density-gradient subcellular fractionation technique to examine the distribution of lysosomal hydrolases between intermediate vesicles (primary lysosomes) and secondary lysosomes in contact-inhibited non-proliferating vs proliferating chicken embryo fibroblasts. We find that the activities of lysosomal specific enzymes from both phases of growth are distributed within two peaks; however, the relative amounts differ markedly. In normal, non-proliferating cells approx. 60% of the total activities of cathepsin B, beta-mannosidase, alpha-fucosidase, beta-galactosidase and hexosaminidase is recovered in the heavier density fraction corresponding to secondary lysosomes, while less than 9% of the enzyme activities are recovered in the light-density peak. With transformed cells, between 16 and 22% of activity for these enzymes are recovered in the lighter density intermediate vesicle fraction, when less than 40% of the enzyme activities recovered in the heavy density fraction. beta-Glucuronidase distribution was different from that of the above enzymes. First, a more even distribution between the two lysosomal fractions was found with non-proliferating normal cells (33% in heavy-density fraction and 21% in light-density fraction), whereas more than 40% of the total enzyme activity was recovered in the lighter density fraction from transformed cells. Also, the amount of cathepsin B contained in the vesicle fractions is increased severalfold relative to that of contact-inhibited normal cells. However, the apparent differences in enzyme distribution between confluent normal and transformed cells are not found when vesicles are prepared from subconfluent, actively proliferating cultures. We have also compared the Percoll density gradient patterns of membrane vesicles from proliferating and non-proliferating human fibroblasts, since most earlier studies utilized this system. Again, we find that the majority of beta-hexosaminidase activity (41%) of contact-inhibited, confluent cells is recovered in the heavier density fraction with less than 15% in the lighter density fraction. Also, the distribution of beta-hexosaminidase between the heavy density and light density vesicle fractions is altered in homogenates from exponentially growing cells, being 22% and 26% respectively. We conclude that the distribution of lysosomal hydrolases between the two vesicle populations is growth-phase dependent and is markedly heterogeneous in proliferating cells.

Animals↗
PubMed1992

Studies of early hepatocellular proliferation and peroxisomal proliferation in Sprague-Dawley rats treated with tumorigenic doses of clofibrate.

Clofibrate, a peroxisome proliferator, is hepatocarcinogenic in rats in a dose-dependent fashion. While there is a relationship between peroxisome proliferation and rodent liver carcinogenesis, recent evidence also suggests an association between the tumorigenicity of peroxisome proliferators and sustained cell proliferation. To investigate the role of early cell proliferation in clofibrate-induced carcinogenesis and the predictive potential of this endpoint, in a 3-month study, rats were fed clofibrate doses equivalent to those used in the chronic bioassay, and cell proliferation was determined after 1 week and 3 months, using a 1-week continuous bromodeoxyuridine (BrdU)-labeling technique. Adult Sprague-Dawley rats were fed clofibrate at 1500, 4500, or 9000 ppm. Six rats/sex/group were killed after 1 or 13 weeks of treatment. Osmotic minipumps containing BrdU were implanted into rats 7 days prior to necropsy to determine the cumulative 7-day hepatocyte labeling index immunohistochemically. A dose-related increase in hepatocyte labeling index was seen after 1 week of treatment. However, at 13 weeks, sustained increases in hepatocyte proliferation were not seen; but a dose-related decrease in the hepatocyte labeling index was observed. Liver stereology at 13 weeks demonstrated a dose-related increase in liver weight and volume, but a decrease in hepatocyte nuclei per unit volume, a minimal increase or no change in the total number of hepatocyte nuclei per liver, and an absolute decline in the total number of BrdU-labeled hepatocyte nuclei per liver. These data suggest that in rats, clofibrate may influence hepatocarcinogenicity by decreases in normal hepatocyte proliferation over time and this effect may influence the pathogenesis of tumors at time points beyond 13 weeks of treatment.

Administration, Oral↗
PubMed1996

Crosstalk between the thyroid hormone and peroxisome proliferator-activated receptors in regulating peroxisome proliferator-responsive genes.

Peroxisome proliferators and thyroid hormones have overlapping metabolic effects and regulate a similar subset of genes involved in maintaining lipid homeostasis. Transcriptional activation by peroxisome proliferators is mediated by peroxisome proliferator-activated receptors (PPARs) that bind to specific peroxisome proliferator-response elements (PPREs) through heterodimerization with retinoid X receptors (RXRs). We examined the effect of thyroid hormone receptor alpha (TR alpha) on DNA binding in vitro and transcriptional activation in vivo by rat PPAR. Gel mobility shift assays using in vitro translated receptors demonstrated that TR alpha was capable of binding on its own and cooperatively with RXR alpha to the rat acyl-CoA oxidase PPRE and of inhibiting the binding of rat PPAR/RXR alpha heterodimers to this element. This inhibition was the result of competition between TR alpha and PPAR for limiting amounts of the heterodimerization partner RXR alpha and for binding to the PPRE. Interestingly, cotransfection of a TR alpha expression plasmid into mammalian cells resulted in potentiation of the peroxisome proliferator- and PPAR/RXR alpha-dependent transcriptional induction of a reporter gene containing the acyl-CoA oxidase PPRE. TR alpha therefore appears to cooperate with RXR and PPAR to positively modulate peroxisome proliferator-dependent transactivation in vivo. Our findings suggest that there is crosstalk between the thyroid hormone and peroxisome proliferator signaling pathways in the regulation of peroxisome proliferator-responsive genes.

Acyl-CoA Oxidase↗
PubMed1991

Efficacy of i.v. or i.p. injected cytotoxic drugs on proliferating and non-proliferating hair follicles of the mouse.

A range of drug doses of Adriamycin (ADR), Actinomycin-D (ACT-D), and Mitomycin-C (MMC) were given i.v. or i.p. to mice 1 day after a priming dose of radiation to elicit epilation response. The hair follicles were stimulated through plucking 11 days before irradiation or were unstimulated to represent proliferating and nonproliferating populations. The maximum epilation that appears at 8 days or at 8.5 weeks for proliferating and nonproliferating follicles was quantified using a subjective scale. In general, the i.v. route of administration was more effective than i.p. for all three drugs. Proliferating follicles were more susceptible than non-proliferating follicles to the action of drugs, especially ADR (p = .0001). Radiation doses which would give the same effect as ADR were calculated for proliferating follicles: 8 mg/kg given i.v. was equivalent to 4.6 (3.9, 5.2) Gy. For i.p. administration, 8 mg/kg was equivalent to only 0.6 (-0.1, 1.3) Gy. The in vivo assay of drug effect on hair follicles has advantages over LD10 as a model for toxicological investigation of new drugs: it can assess response of proliferating or non-proliferating cells of the same histotype and, in the case of proliferating follicles, it is quicker, thus enabling the use of doses higher than LD50 for bone marrow deaths.

Animals↗
PubMed2001

Human peroxisome proliferator-activated receptor alpha (PPARalpha) supports the induction of peroxisome proliferation in PPARalpha-deficient mouse liver.

Peroxisome proliferators, which function as peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, induce peroxisomal, microsomal, and mitochondrial fatty acid oxidation enzymes, in conjunction with peroxisome proliferation, in liver cells. Sustained activation of PPARalpha leads to the development of liver tumors in rats and mice. The assertion that synthetic PPARalpha ligands pose negligible carcinogenic risk to humans is attributable, in part, to the failure to observe peroxisome proliferation in human hepatocytes. To explore the mechanism(s) of species-specific differences in response to PPARalpha ligands, we determined the functional competency of human PPARalpha in vivo and compared its potency with that of mouse PPARalpha. Recombinant adenovirus that expresses human or mouse PPARalpha was produced and administered intravenously to PPARalpha-deficient mice. Human as well as mouse PPARalpha fully restored the development of peroxisome proliferator-induced immediate pleiotropic responses, including peroxisome proliferation and enhanced expression of genes involved in lipid metabolism as well as nonperoxisomal genes, such as CD36, Ly-6D, Rbp7, monoglyceride lipase, pyruvate dehydrogenase kinase-4, and C3f, that have been identified recently to be up-regulated in livers with peroxisome proliferation. These studies establish that human PPARalpha is functionally competent and is equally as dose-sensitive as mouse PPARalpha in inducing peroxisome proliferation within the context of mouse liver environment and that it can heterodimerize with mouse retinoid X receptor, and this human PPARalpha-mouse retinoid X receptor chimeric heterodimer transcriptionally activates mouse PPARalpha target genes in a manner qualitatively similar to that of mouse PPARalpha.

Animals↗
PubMed1998

Proliferation and cellular phenotype in lymphomatoid granulomatosis: implications of a higher proliferation index in B cells.

Pulmonary involvement by lymphomatoid granulomatosis (LYG) is characterized by nodules of a polymorphous lymphoreticular infiltrate with necrosis, angioinvasion, and variable numbers of large, atypical cells. Using combined immunohistochemistry, the authors compared the expression of a marker of proliferation (DNA topoisomerase IIalpha) between B cells, T cells, and histiocytes. Sixteen cases of LYG were stained by combined immunohistochemistry for DNA topoisomerase IIalpha and CD-20, CD-3, CD-68, and CD-57. A proliferation index was determined for B cells, T cells, histiocytes, and natural killer cells by dividing the number of cells with coexpression of DNA topoisomerase IIalpha and CD-20, CD-3, CD-68, or CD-57 by the total number of CD-20+, CD-3+, CD-68+, or CD-57+ cells, respectively. A significantly higher proliferation index was present in B cells compared to T cells, histiocytes, or natural killer cells (p < 0.002). The average proliferation index for B cells was 0.25+/-0.24 (range, 0.00-0.76), for T cells was 0.02+/-0.01 (range, 0.00-0.04), for histiocytes was 0.00+/-0.01 (range, 0-0.02), and for natural killer cells was 0.00+/-0.00 (range, 0.0-0.02). The average proliferation index of CD-20+ cells was greater in grade III LYG (0.36) than in grade II LYG (0.17) or the single case of grade I LYG (0.00). The authors conclude that (1) there is a spectrum of B-cell proliferation in LYG that roughly correlates with histologic grade, (2) T cells, histiocytes, and natural killer cells do not proliferate but are recruited, and (3) the average B-cell proliferation index in grade III LYG is similar to that observed in large cell non-Hodgkin's B-cell lymphomas. These observations provide a possible rationale for the use of chemotherapy for grade III LYG and observation or immunologic adjuvants for LYG with grade I or grade II histology.

Adult↗
PubMed1992

Inhibition of lung epithelial cell proliferation by hyperoxia. Posttranscriptional regulation of proliferation-related genes.

The alveolar surface of the lung is a major target for oxidant injury. After injury, repair of the alveolar epithelium is dependent on the ability of epithelial type 2 (T2) cells to proliferate. The regulation of T2 cell proliferation and the effect of reactive oxygen (O2) species on this lung cell proliferation have not been well defined. To investigate this process we focused on the regulation of two late cell cycle genes, histone and thymidine kinase, in T2 cells and fibroblasts exposed in vitro to varying periods of hyperoxia (95% O2). Hyperoxia for 24 to 48 h arrested cell proliferation in a SV40T-immortalized T2 cell line we have developed and in primary and SV40T-immortalized lung fibroblasts. Despite the cessation of proliferation, histone and TK mRNA continued to be expressed at high levels; mRNA half-lives were markedly prolonged but neither protein was translated. Thus proliferation arrest induced by hyperoxia was associated with posttranscriptional control of at least two late cell cycle-related genes. This form of proliferation arrest is also seen when primary and SV40T-T2 cells but not fibroblasts are serum deprived, suggesting that T2 cells in vitro may be uniquely sensitive to alterations in their redox state and that these alterations in turn affect translational control of a subset of proliferation-related genes.

Animals↗
PubMed1995

A novel single-cell proliferation assay shows that long-term culture-initiating cell (LTC-IC) maintenance over time results from the extensive proliferation of a small fraction of LTC-IC.

We have previously shown that when adult marrow CD34+/HLA-DR- cells are cultured for 5 or 8 weeks in the presence of stroma-conditioned media with interleukin-3 (IL-3) and macrophage inflammatory protein-1 alpha (MIP-1 alpha), long-term culture-initiating cells (LTC-IC) are maintained but not expanded. However, if the same cultures are evaluated after 2 weeks, we show that LTC-IC expand 5.5- +/- 0.2-fold. Because expansion of LTC-IC is likely the result of a balance between proliferation and loss of LTC-IC, we hypothesized that, although LTC-IC proliferate in these cultures, loss of a fraction of LTC-IC underlies the lack of long-term expansion. To evaluate the fate of LTC-IC (proliferation, conservation, or loss), we performed PKH-26 labeling assays and developed a single LTC-IC proliferation assay. For PKH-26 labeling assays, CD34+/HLA-DR- cells were incubated with the membrane intercallating dye, PKH-26, before culture for 14 days in stroma-noncontact cultures + IL-3 + MIP-1 alpha. Progeny was reselected by fluorescence-activated cell sorting based on their PKH-26 fluorescence intensity. These studies showed that LTC-IC proliferate because 80% of LTC-IC at week 2 had 0.5 to 1 log lower fluorescence intensity than did freshly labeled CD34+/HLA-DR- cells. To further determine the fate of LTC-IC, we also developed a single LTC-IC proliferation assay. A population of CD34+/CD33- cells, highly enriched in LTC-IC, was sorted singly in stroma-conditioned media+IL-3 + MIP-1 alpha. After 5 weeks, the content of each well was divided equally over 8 secondary stroma-containing wells and cultured for 8 weeks to determine the capacity of the single-cell progeny to initiate 1 or more secondary stromal cultures. Progeny of single-sorted cells were able to initiate up to 8 secondary long-term cultures, demonstrating that LTC-IC proliferate in stroma-conditioned media+IL-3 + MIP-1 alpha. However, more than 65% of single-sorted LTC-IC were not conserved because their progeny could no longer initiate secondary long-term cultures. This finding indicates that, although stromal factors and IL-3 + MIP-1 alpha can induce proliferation of LTC-IC, failure to conserve a large fraction of LTC-IC results in lack of long-term expansion.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1996

Hepatocyte proliferation induced by a single dose of a peroxisome proliferator.

In compensatory hyperplasia after partial hepatectomy or liver cell injury, hepatocyte proliferation is triggered by coordinated actions of growth factor such as hepatocyte growth factor and transforming growth factor-alpha and -beta. Initiation of hepatocyte DNA synthesis is preceded by the activation of the set of early growth response genes mediated by enhanced nuclear factor-kappa B binding to DNA. Using an experimental model to induce hepatocyte DNA synthesis in vivo by a single dose of a peroxisome proliferator, which does not induce liver cell necrosis (direct hyperplasia), we investigated whether peroxisome proliferator-induced hepatocyte proliferation involved an induction of known growth factors, an activation of early growth response genes, and nuclear factor-kappa B. A single intragastric administration of 250 mg/kg BR931 (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide) to male wistar rats induced a wave of hepatocyte DNA synthesis starting after 12 hours and peaking at approximately 24 to 36 hours. The response was dose dependent. The treatment also induced the expression of the mRNA for the peroxisomal bifunctional enzyme, one of the peroxisome-related fatty acid beta-oxidation enzymes. Pretreatment of rats with dexamethasone (2 mg/kg) inhibited both hepatocyte DNA synthesis and the induction of the peroxisomal bifunctional enzyme gene. Northern blot analyses of liver RNA during a period preceding the onset of DNA synthesis revealed no induction of hepatocyte growth factor, transforming growth factor-alpha, or tumor necrosis factor-alpha mRNAs. No induction of early growth response genes, liver regeneration factor-1, or c-myc was detected. Furthermore, gel mobility shift assays showed no enhanced nuclear factor-kappa B binding to its DNA consensus sequence after BR931 treatment, whereas control studies demonstrated a distinct increase in binding after partial hepatectomy or lead nitrate treatment. The results suggest that peroxisome-proliferator-induced hepatocyte proliferation may be triggered by signal transduction pathways different from those after partial hepatectomy and that the binding of peroxisome proliferators to their nuclear receptors may play a role in stimulation of DNA synthesis and peroxisome proliferation.

Animals↗
PubMed1994

Local proliferation of fibroblast-like synoviocytes contributes to synovial hyperplasia. Results of proliferating cell nuclear antigen/cyclin, c-myc, and nucleolar organizer region staining.

OBJECTIVE: To test the hypothesis that local proliferation contributes significantly to the hyperplasia of rheumatoid synovium. METHODS: Immunohistologic and chemical staining was used to identify 3 markers of cell proliferation: proliferating cell nuclear antigen, c-myc proto-oncogene, and nucleolar organizer regions. Synovium from 21 patients with rheumatoid arthritis, 34 with degenerative joint disease, and 7 with joint trauma was examined. RESULTS: All 3 markers indicated substantial, active proliferation of synovial lining cells in synovium with hyperplasia. Proliferating cells showed type I procollagen immunoreactivity but were negative for CD68, a monocyte/macrophage marker. Proliferation was greater in rheumatoid arthritis than in the other conditions evaluated. CONCLUSION: In situ proliferation of fibroblast-like synoviocytes in the synovium lining contributes considerably to the increase in cell numbers in rheumatoid synovium.

Adult↗
PubMed2003

Cell proliferation and differentiation during fracture healing are influenced by locally applied IGF-I and TGF-beta1: comparison of two proliferation markers, PCNA and BrdU.

Growth factors IGF-I and TGF-beta1 are known to stimulate fracture healing. The purpose of this study was to investigate the role of locally applied IGF-I and TGF-beta1 during the early phase of fracture healing (Days 5, 10, and 15 after fracture) on cellular processes like proliferation and differentiation in a rat model. Two different immunohistochemical markers were used to analyze cell proliferation: (1) injection of the thymidine analogue BrdU and subsequent immunohistochemical staining for BrdU-positive nuclei, and (2) the antibody against the "proliferating cell nuclear antigen" (PCNA). In comparison, both methods revealed similar results concerning the types of proliferating cells at the different time points and the two groups. Labeling indices of both methods showed very good correlation (e.g., r(s): 0.887 and p < 0.001 at day 10 in the control group without growth factors). Comparison of the callus morphology and the proliferation rate showed differences during fracture healing due to the local application of IGF-I and TGF-beta1 from coated implants. At Day 5 the callus of the group treated with growth factors displayed an earlier appearance of cartilage compared to the control group. This was accompanied by an onset of cell proliferation in chondrocytes. Likewise, at the later time points an enhanced maturation of the callus tissue and the proliferation pattern were detectable in the growth-factor group. These results indicate that local application of IGF-I and TGF-beta1 accelerates early cellular processes during fracture healing.

Animals↗