Cell Replication Rates Research Articles

Effects of whey-milk proteins on Caco-2 and HT-29 intestinal cell lines

Abstract The objective of this study was to determine whether α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) affect proliferation of intestinal cells. As a consequence of intake of milk, epithelial cells of the intestine may be subjected to small concentrations of undergraded milk proteins. The question was whether α-LA or β-LG have growth promoting or inhibiting properties in the intestinal epithelial cell model systems, Caco-2 and HT-29. Cells were grown as recommended by ATCC. Native bovine α-LA, or 2 and 4% trypsin hydrolyzed α-LA were added to cultures of Caco-2 cells in dosages of 0 to 50 ng/mL. After 24 to 120 h of culture, cell proliferation assays were done. By using the XTT cell proliferation assay, by visually counting cells in selected microscope fields, and by counting trypsinized cell suspensions in a hemocytometer, α-LA consistently increased cell replication rate with a peak response at approximately 20 ng/mL. For example, the HTT assay showed 85% increased mitochondrial dehydrogenase activity in the 15 ng/ml treatment relative to control. Higher concentrations were either less effective or ineffective growth stimulators in both cell lines. At 2% α-LA hydrolysis, there was a slight stimulation (7%) and no effect at 4% hydrolysis. There was no response using bovine β-LG as supplement. Variance from plating to plating was high, but percent changes from dose to dose within a plate were consistent over the replicates (C.V.

EFFECTS OF EXPOSURE TO ELECTROMAGNETIC RADIATION AT 835 MHz ON GROWTH, MORPHOLOGY AND SECRETORY CHARACTERISTICS OF A MAST CELL ANALOGUE, RBL-2H3

A mast cell line, RBL-2H3, was exposed to 835MHz for 20 minutes, three times per day for 7 days at a power density of 8.1±3mW/cm2From day 4 onwards, it was observed that the rate of DNA synthesis and cell replication increased, that actin distribution and cell morphology became altered, and the amount of β-hexosaminidase (a marker of granule secretion) released in response to a calcium ionophore was significantly enhanced, in comparison to unexposed cultures. There were no effects seen on levels of cytoskeletal protein synthesis or of beta-actin mRNA. Morphological changes persisted following subculture for at least 7 days in the absence of further exposure. It is hypothesized that effects of exposure to an electromagnetic field at 835MHz may be mediated via a signal transduction pathway.

Osteoarthritis in the temporo-mandibular joint (TMJ) of aged mice and the in vitro effect of TGF-beta 1 on cell proliferation, matrix synthesis, and alkaline phosphatase activity.

The temporo-mandibular joint of aged mice develops osteoarthritic (OA) degenerative lesions. Adult chondrocytes have a low rate of cell replication, and cartilage repair potential is very limited. One of the major problems in OA is the low rate of matrix synthesis and the inability of the chondrocytes to exceed the rate of matrix degradation. These combined factors lead to the overall destruction of the cartilage as seen in OA. Cartilage degradation is mediated by elevated proteolytic activity of enzymes. Among the enzymes degrading cartilage are the metalloproteinases, stromelysin and collagenase. Other proteinases that may potentially participate in matrix degradation are the lysosomal enzymes cathepsin B, D, and L, and acid phosphatase. On the other hand, alkaline phosphatase (ALP) is an enzyme that has been shown to be a marker for anabolic activity in skeletal tissues such as bone and cartilage. The cartilage of the mandibular condyle in the T-M-J from aged mice reveals OA lesions. An overall reduction of cell proliferation and sulfated proteoglycan synthesis has been also shown in this joint. In the present study the effects of hTGF-beta on the stimulation of DNA and sulfated GAG synthesis and ALP activity were studied. Mandibular condyle cartilage obtained from 12-month-old ICR male mice were cultured in BGJb serum-free medium for 24-72 hours, supplemented with 0.1-10 ng/ml hTGF-beta 1. 3H-thymidine and 35S-sulfate were added for the last 24 hours of the culture and their incorporation into DNA and sulfated GAGs respectively, as well as the activity of ALP, were determined. Results indicated that hTGF-beta 1 enhanced the incorporation of both 3H-thymidine and of 35S-sulfate into cartilage cultures of aged mice, and also induced ALP activity. It thus appeared that in OA degenerating articular cartilage, the chondrocytes could be stimulated in vitro to proliferate and to synthesize new matrix, thus indicating induced anabolic activity in the tissue.

Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regression.

We quantitatively assessed rates of cell replication and of apoptosis during the development and regression of liver cancer. In rats, apoptotic activity gradually increased from normal liver to putative preneoplastic foci (PPF), to hepatocellular adenoma (HCA), and to hepatocellular carcinoma (HCC). At all stages, rates of cell replication were higher than of apoptosis, allowing a preferential net gain of (pre)neoplastic cells. As in rats, in human HCC, birth and death rates were increased manifold, indicating a species-independent phenomenon. Implications of the increasing cell turnover were studied in rats using the administration and withdrawal of nafenopin (NAF), a liver mitogen and nongenotoxic carcinogen. Prolonged NAF treatment enhanced cell number in normal liver by 25%, while PPF and liver tumors were amplified at least 100-fold. After stopping NAF treatment, cell replication ceased, while cell elimination by apoptosis was increased in normal and (pre)neoplastic liver. HCA and HCC showed the most pronounced shifts from replication toward apoptosis. As a result, 5 weeks after halting NAF, 20% of cells in normal liver, but about 85% of (pre)neoplastic lesions including HCC, were eliminated. The implications of these findings include that nongenotoxic carcinogens can act as survival factors even for malignant cells. Furthermore, tumor cells not only exhibit excessive proliferation, but also undergo apoptosis at rates that far exceed those in normal tissue. Therefore, inhibition of cell death by the survival activity of nongenotoxic carcinogens results in selective growth of (pre)neoplastic lesions. On the other hand, blockade of survival effects leads to excessive apoptosis in (pre)neoplasia and seems promising as a therapeutic concept for the selective elimination of (liver) cancer.

Nitric oxide and pancreatic islet blood flow after induced portal hypertension in rats.

Portal hypertension (PH) is associated with a hyperdynamic splanchnic circulation, partially mediated by nitric oxide-dependent mechanisms. The aim of the present study was to evaluate the influence of PH and nitric oxide on pancreatic islet blood flow. PH was induced by a calibrated stenosis of the portal vein in male Sprague-Dawley rats. Control were sham-operated. Ten days later pancreatic, duodenal, colonic, and arterial hepatic blood flows were measured with microspheres. All splanchnic blood flow values were markedly increased in the PH rats. The fraction of whole pancreatic blood flow diverted through the islets increased from approximately 5 to 15%. Intravenous administration of the nitric oxide synthase inhibitor NG-nitro-L-arginine (25 mg/kg body weight) in rats with PH 10 min before blood flow measurements decreased pancreatic, duodenal, colonic, and arterial hepatic blood flow to control values. Pancreatic islet blood flow was also decreased, but more markedly than that of the whole pancreas. Pancreatic islet morphology was normal, and the rate of islet cell replication was not influenced. PH induced a preferential increase in pancreatic islet blood flow, which is likely to be associated with an increased production of nitric oxide.

Neutralizing antibodies directed against osteopontin inhibit rat carotid neointimal thickening after endothelial denudation.

Osteopontin is an arginine-glycine-aspartate-containing acidic glycoprotein with adhesive and migratory activities in vitro. We previously showed that osteopontin was highly expressed in injured rat arteries as well as in human atherosclerotic plaques. In contrast, uninjured blood vessels make very little osteopontin. In this report, we have investigated the role of osteopontin in rat neointima formation using neutralizing antibodies. Rats were treated with either nonimmune or antiosteopontin antibody and subjected to endothelial denudation of the carotid artery by using a balloon catheter. Two weeks after injury, intimal areas and cell numbers were significantly decreased (33% and 31%, respectively) in the antiosteopontin group compared with the nonimmune IgG group. No differences in carotid medial areas or cell numbers were observed. Intimal and medial replication rates, as measured by continuous bromodeoxyuridine infusion during the final week of the experimental protocol, were not significantly different between the two groups. No gross histological changes were noted in the intimas formed in the presence of either neutralizing or nonimmune antibody. In addition, no difference in early carotid medial cell replication rate was observed when antibodies were infused for 4 days after angioplasty. These data demonstrate for the first time a functional role for osteopontin in the process of carotid neointimal thickening in vivo and suggest that osteopontin plays an active role in the remodeling processes important for human atherosclerotic and restenotic lesion development.

Differential expression of alpha 1 type VIII collagen in injured platelet-derived growth factor-BB--stimulated rat carotid arteries.

Migration of smooth muscle cells from media to intima is critical for the development of neointimal thickening after balloon catheter injury of the rat carotid artery. The present experiments were designed to identify molecules expressed by smooth muscle cells migrating in vivo in the injured artery. Cell migration was maximized by infusing recombinant platelet-derived growth factor-BB (PDGF-BB) after a minimal filament denudation of the rat carotid artery, whereas cell proliferation was minimized by injecting an antibody against basic fibroblast growth factor (bFGF). This treatment caused an eightfold increase in smooth muscle cell migration into the intima but only a twofold increase in intimal smooth muscle cell replication rates. Differential display screening was used to isolate cDNAs that were overexpressed in the injured PDGF-BB-treated versus unmanipulated rat carotids. One of the clones isolated hybridized to a 4.2-kb mRNA species and shared 90% sequence homology to mouse alpha 1 type VIII collagen. Northern and Western blots confirmed overexpression of type VIII collagen in the injured PDGF-BB-treated vessels. In a separate series of experiments, we performed filament denudation injury and administered antibodies to inhibit the actions of endogenous bFGF and PDGF-BB, thereby decreasing smooth muscle cell migration, and found that type VIII collagen mRNA expression varied with migration. Using a different arterial injury model (balloon catheter injury), we showed that expression of type VIII collagen was maximal 2 to 4 days after injury, in coincidence with cell migration from the media to the intima. This molecule constitutes an important component of smooth muscle cell response to vessel injury and may play an important functional role in mediating migration.

Nonclinical Toxicology Studies with Zidovudine: Acute, Subacute, and Chronic Toxicity in Rodents, Dogs, and Monkeys

In single dose acute toxicity studies in CD-1 mice and CD rats, the median lethal dose (MLD) for zidovudine (ZDV) was >750 mg/kg after iv dosing and >3000 mg/kg after po administration (recommended human dose is 100 mg every 4 hr while awake). Because of the short half-life in rats (0.8 hr), dogs (1.0 hr), and monkeys (0.8 hr), the daily dose of ZDV in most studies was given in two equal portions approximately 6 hr apart. Intravenous administration of ZDV was well tolerated in beagle dogs at dose levels up to 42.5 mg/kg bid for 2 weeks and in CD rats at dose levels up to 75 mg/kg bid for 4 weeks. In a 2-week dose range-finding study in beagle dogs, cytostatic effects were noted at po dose levels of 62.5 to 250 mg/kg bid in certain tissues with rapid cell replication rates. In contrast, in 3- to 12-month oral toxicity studies in CD rats and cynomolgus monkeys, the principal toxicologic finding was reversible macrocytic normochromic anemia which occurred at 225–250 mg/kg bid in rats and 17.5–150 mg/kg bid in monkeys. In the 12-month rat study, RBC was decreased at 25 and 75 mg/kg bid. In the 12-month monkey study WBC was slightly decreased at 150 mg/kg bid.

Incorporation of bromodeoxyuridine in regenerating fin tissue of the goldfish Carassius auratus.

We have investigated the pattern of incorporation of 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU) by proliferating cells during regeneration of the tail fin of Carassius auratus. Fifteen days after amputation, intraperitoneal injection of a single dose of 0.25 mg/g wet weight of BrdU and subsequent immunocytochemical detection on sections revealed groups of replicating cells in the blastema and epidermis at different proximodistal levels. Proliferating blastemal cells were confined to a crowded, compact distal area that lost its replicative capacity laterally, causing the differentiation of scleroblasts, which synthesize the lepidotrichia hemisegments. Proximally, but centrally located, the blastemal cells did not incorporate BrdU and they differentiated giving rise to the mature intraray connective tissue. An independent cell-proliferation process was noted in the epidermis. The distal cap did not proliferate; the lateral faces of the epidermis showed high rates of cell replication in the central layer at every level of the regenerate rays; quiescent cells remained in the superficial layers. The basal epidermal cells did not incorporate BrdU when actinotrichia were present. The possible role of basal epidermal cells in the synthesis of actinotrichia, the contribution of these collagen macrofibrils to the morphogenetic process, and the different pathways of cell differentiation during fin regeneration are discussed.

Apoptosis and vascular wall remodeling in hypertension

Vascular structure plays a key role in the pathogenesis of hypertension. Because it is less readily reversible than protein accumulation, DNA accumulation in the arterial wall may be considered as a record of past episodes of vascular growth, contributing to the persistence of the vascular disease. Apoptosis, a ubiquitous and highly regulated form of programmed cell death that is involved in tissue morphogenesis and homeostasis as the essential counterpart of cell replication, is potentially involved in the regulation of vascular structure, via the deletion of cells in the vessel wall. We discuss how the current knowledge on apoptosis may provide insights into the pathogenesis of vascular wall remodeling, with an emphasis on the biology of vascular smooth muscle cells. Evidence suggests that heightened cell replication rates are often associated with increased apoptosis, as for smooth muscle cells in genetic hypertension or in arterial repair after injury. However, apoptotic cell death may also be regulated independently of cell growth. The triggering of apoptosis depends on a balance of environmental cues that are not specific to apoptosis. However, there is a common set of key biochemical events mediating apoptosis (i.e., committing the cell to die), thus providing a basis for the design of novel pharmacological strategies specifically targeting apoptotic cell death. The identification of biochemical markers of apoptosis and other methodological advances will ultimately help in understanding the role of apoptotic cell death in vascular remodeling and hypertensive end-organ damage.

Nonclinical Toxicology Studies with Zidovudine: Acute, Subacute, and Chronic Toxicity in Rodents, Dogs, and Monkeys

In single dose acute toxicity studies in CD-1 mice and CD rats, the median lethal dose (MLD) for zidovudine (ZDV) was > 750 mg/kg after iv dosing and > 3000 mg/kg after po administration (recommended human dose is 100 mg every 4 hr while awake). Because of the short half-life in rats (0.8 hr), dogs (1.0 hr), and monkeys (0.8 hr), the daily dose of ZDV in most studies was given in two equal portions approximately 6 hr apart. Intravenous administration of ZDV was well tolerated in beagle dogs at dose levels up to 42.5 mg/kg bid for 2 weeks and in CD rats at dose levels up to 75 mg/kg bid for 4 weeks. In a 2-week dose range-finding study in beagle dogs, cytostatic effects were noted at po dose levels of 62.5 to 250 mg/kg bid in certain tissues with rapid cell replication rates. In contrast, in 3- to 12-month oral toxicity studies in CD rats and cynomolgus monkeys, the principal toxicologic finding was reversible macrocytic normochromic anemia which occurred at 225-250 mg/kg bid in rats and 17.5-150 mg/kg bid in monkeys. In the 12-month rat study, RBC was decreased at 25 and 75 mg/kg bid. In the 12-month monkey study WBC was slightly decreased at 150 mg/kg bid.

Apoptosis contributes to the involution of beta cell mass in the post partum rat pancreas.

A significant reduction of beta cell mass has been described during the post partum period in the endocrine rat pancreas. We examined the mechanisms of this involution in Sprague Dawley rats by analyzing beta cell mass, beta cell replication, and beta cell size at end of pregnancy and 4 and 10 days after delivery. beta cell replication was significantly decreased at 4 days post partum but had returned back to nonpregnant levels by 10 days post partum. Similarly, beta cell size was significantly decreased at 4 and 10 days post partum as compared with the end of pregnancy, and at 10 days post partum was significantly decreased as compared with controls. At 4-6 days post partum, DNA fragmentation characteristic of apoptosis (programmed cell death) was detected in pancreatic islets, as assessed by in situ terminal deoxynucleotidyl transferase and nick translation assay. Only occasional cells were labeled with this assay in nonpregnant rats and at other time points after delivery. Condensed chromatin and apoptotic bodies, the morphological characteristics of apoptosis, were detected in beta cells of pancreatic islet at 3 and 4 days after delivery by electron microscopic analysis, confirming the occurrence of apoptosis in involuting islets. The expression of TRPM 2 and TGF beta 1, often enhanced in models of apoptosis, were studied during the post partum period by Northern blot analysis and immunohistochemistry. Levels of TRPM 2 gene and its protein, clusterin, were not different from controls; however, the TGF beta 1 gene and its protein expression were enhanced at 3 days post partum. Our study confirms the capability of beta cells to down-regulate their mass using the mechanisms of changes in rates of beta cell replication and of beta cell death, and changes in beta cell size to achieve homeostasis of the functional endocrine tissue.

Evolutionary consequences of mutation and selection within an individual.

Whether in sexual or asexual organisms, selection among cell lineages during development is an effective way of eliminating deleterious mutations. Using a mathematical analysis, we find that relatively small differences in cell replication rates during development can translate into large differences in the proportion of mutant cells within the adult, especially when development involves a large number of cell divisions. Consequently, intraorganismal selection can substantially reduce the deleterious mutation rate observed among offspring as well as the mutation load within a population, because cells rather than individuals provide the selective "deaths" necessary to stem the tide of deleterious mutations. The reduction in mutation rate among offspring is more pronounced in organisms with plastic development than in those with structured development. It is also more pronounced in asexual organisms that produce multicellular rather than unicellular offspring. By effecting the mutation rate, intraorganismal selection may have broad evolutionary implications; as an example, we consider its influence on the evolution of ploidy levels, finding that cell-lineage selection is more effective in haploids and tends to favor their evolution.

Effect of mechanical forces on growth and matrix protein synthesis in the in vitro pulmonary artery. Analysis of the role of individual cell types.

The effect of mechanical stimuli on pulmonary artery growth and matrix tissue synthesis (and how individual cell types in the vessel wall respond to such stimuli) is incompletely characterized. Rabbit pulmonary arteries were placed in tissue culture medium and subjected to varying magnitudes of stretch or hydrostatic pressure (separately) for 4 days. The rate of protein synthesis in smooth muscle cells (by quantitative autoradiography) was positively related to the magnitude of stretch, as were the percentage of procollagen type I-positive cells and the rate of cell replication. In adventitial fibroblasts, stretch increased the rate of replication but not of protein synthesis. Hydrostatic pressure had little or no effect on the variables measured in either smooth muscle cells or fibroblasts. Stretch also increased the rate of elastin and collagen synthesis in the whole pulmonary artery segment, and after 4 days of stretch, the contents of actin and elastin were increased. Removal of the endothelium did not affect stretch-induced protein, collagen, or elastin synthesis but augmented stretch-induced smooth muscle replication. These data suggest that in the intact pulmonary artery, stretch, but not pressure, can stimulate hypertrophy and hyperplasia in smooth muscle cells and hyperplasia in fibroblasts. Matrix protein synthesis and accumulation are also increased by stretch. Neither stretch-mediated growth nor matrix protein synthesis required endothelium in this model.

Rates of growth of human solid neoplasms: Part I.

The purpose of this article is to consolidate data collected from a variety of sources that have permitted calculations of the rates of growth of human neoplasms. These sources include Fischel State Cancer Hospital (Columbia, MO); Mallinckrodt Institute of Radiology, (St. Louis, MO); Roentgen Diagnostic Institute, Allmanna Sjukhuset (Malmo, Sweden); University of Louisville (Louisville, Kentucky); University of Heidelberg (Heidelberg, Germany); and St. Luke's Hospital (St. Louis, MO). Included in the data are laboratory measurements of cell replication rates. All gross measurements were made either on imaging studies or with a centimeter scale for surface or palpable neoplasms. Data have been reported for breast and pulmonary cancers and metastases of many types, melanomas, skeletal sarcomas, benign and malignant colonic neoplasms, and isolated cases of less frequent neoplasms. Related cytokinetic measurements by tritriated thymidine labelling, bromodeoxyuridine labelling, S-phase fraction from DNA flow cytometric analysis, and mitotic indices are discussed. The various mathematical formulae applicable to the analysis of the collected data and the determination of rates and patterns of growth are included. Also considered are the clinical implications of these data and the importance of ever better knowledge on the cytokinetics of human cancer. Prior studies on the evolution of insight into this field are cited and discussed. The authors conclude that a more accurate quantification of the growth rates of human cancer is essential for understanding the biological variance of human cancers seen clinically.

Caloric Restriction: Conservation of in Vivo Cellular Replicative Capacity Accompanies Life-Span Extension in Mice

In male mice of a long-lived hybrid strain (B6D2F1), long-term 40% caloric restriction (CR) extended both mean and maximum life spans by 36 and 20%, respectively, over that of ad libitum fed (AL) controls. Measurements of entry into S-phase were made in vivo of six different cell types in five different organs using 2-week exposures to BrdU. The labeling index (L.I.) in all organs studied was lower in young CR mice than in young AL fed mice. In most cases, the L.I. in AL mice fell to the levels of that in the CR mice by 13 months of age, and the two groups then remained so through old age. However, when the L.I. was measured in old CR mice which had been placed on the AL diet for a period of 4 weeks (this was termed refeeding (RF)), it was found to be above that of similar age AL or CR mice and almost at the level of young AL mice. This was still true, but to a lesser degree, in a repeat study using an 8-week period of RF. In a separate but parallel in vitro study (companion paper, this volume), the superiority of CR over AL for retention of cellular replication capacity was confirmed by clone size distribution measurements made in several cell types in mice of several age groups. These results indicate that: (1) the rate of cell replication in AL diet mice diminishes greatly by early middle age in all organ sites studied and then plateaus or declines much more slowly; (2) CR broadly preserves in vivo cellular replicative capacity but often requires the energy levels provided by a switch to AL feeding to demonstrate this late in life; (3) accordingly, the replicative deficit in AL fed mice appears to be cumulative and is significant only in old age. The mechanism(s) involved is yet to be discovered but may be related to, or even the same as, that which extends life spans in CR animals. Correspondingly, and with corroborative data from our in vitro companion study, (W. R. Pendergrass et al., 1995. Exp. Cell. Res. 217, 309-316), we suggest that cell populations sustain an accrual of biochemical damage or physiological alterations which increasingly limit their replicative capacity as the animal ages, and that CR reduces the accrual of this damage.

Caloric Restriction: Conservation of Cellular Replicative Capacity in Vitro Accompanies Life-Span Extension in Mice

We have tested whether life-long caloric restriction (CR) slows or delays the age-related loss of cellular replicative potential that occurs during normal aging in ad libitum (AL) fed mice. Both mean and maximum life spans of the restricted animals (60% of AL intake) were significantly extended 30-40% by CR treatment. Proliferative potential, measured by determining the fraction of cells capable of forming large clones in vitro, was compared in five cell types from six tissue sites from two strains of mice (Male (C57BL/6 × DBA/2)F1("B6D2F1") and female (C57B1/6 × C3H)F1("B6C3F1")). This included four nonhematopoietic organ sites: fibroblast cells from ear skin, tail skin, and subdermal connective tissue and epithelial cells from the medullary part of the kidney and two cell types, myofibroblasts and endothelial-like cells, from spleen and bone marrow. The proliferative potential of cells from AL mice decreased progressively with age in all tissues sites of both mouse strains. CR delayed or decreased the loss of proliferative potential in all situations, but the timing of this was tissue specific. For cells from the four nonhematopoietic tissue sites from female B6C3F1 female mice, CR delayed the onset of proliferative loss, such that the fraction of large clones was significantly greater for the CR 18- to 24-month-old mice than in AL controls at three of four sites (as determined by the fraction of large clones after 1 week of clonal growth). The proliferative loss in CR tissues then accelerated from 24 to 30 months, so that both CR and AL mice had similar fractions of large clones after 30 months of age. CR was also seen to delay loss of proliferative potential in cells from skin and kidney of B6D2F1 male mice at 23-24 months of age when cloned for 2 weeks. For fibroblast and endothelial-like cells from bone marrow and spleen stromal sites from both strains of mice, CR also significantly decreased loss of proliferative potential; furthermore, in these tissues the proliferative advantages remained or increased from 24 to over 30 months of age. In companion studies (N. S. Wolf et al., 1995. Exp. Cell. Res. 217, 000-000), CR was seen to decrease age-related losses in the maximal rates of cell replication in vivo in a panel of tissues from B6D2F1 male mice. The preservation of replicative potential by CR mice in all tissues tested, both in vitro and in vivo, indicates that CR preserves proliferative capacities in the cells and tissues of chronically restricted mice and may permit CR mice to better respond to proliferative stresses in old age.

Growth abnormalities in cultured mesangial cells from rats with spontaneous glomerulosclerosis

Age-related glomerulosclerosis (GS) occurs in normotensive rats of the Milan strain (MNS), but not in genetically-matched hypertensive animals (MHS). Altered mesangial cell (MC) proliferation and matrix expansion are common features of the glomerular scarring process. We evaluated proliferation and matrix protein synthesis of cultured MC from MNS and MHS animals aged 1 and 8 months, that is, before and after the occurrence of GS. [3H]-thymidine (TdR) incorporation into DNA of MC from MNS rats stimulated by 10% FBS serum increased with donor aging from 115 +/- 6.0 to 176 +/- 15, P < 0.01 (% cpm/well over quiescent controls +/- SEM). Under the same experimental conditions, cell counts changed from 101 +/- 4.0 to 146 +/- 5.0, P < 0.01 (% cells/well over quiescent controls). Additionally, cytosolic Ca2+ concentration ([Ca2+]i) rised from 115 +/- 19 to 220 +/- 32 nM and from 112 +/- 24 to 734 +/- 136 nM when fura-2-loaded cells from young and old MNS rats, respectively, were stimulated with 1% FBS. The rate of collagen production also increased with donor age, as well as collagen IV and laminin B1 mRNA expression. In contrast, in MC from MHS rats both DNA synthesis and cell replication rate declined as function of donor age. No differences in the [Ca2+]i responses to FBS were observed, nor collagen production changed with MHS rat senescence. We conclude that the age-associated decline of proliferative activity in MC from MHS animals could actually reflect a normal process of cell aging, possibly protecting from the occurrence of GS.(ABSTRACT TRUNCATED AT 250 WORDS)

Active cell death: role in hepatocarcinogenesis and subtypes.

Active cell death, the genetically programmed self-destruction of a cell, is now recognized to be a widespread phenomenon in biology that counterbalances mitosis to preserve tissue homeostasis. It is subject to the control of the growth regulatory networks in tissues. Close examination of the morphology of dying cells in liver and other tissues suggests that there are a number of morphological types of active cell death, ranging from forms dominated by nuclear changes and without signs of autophagy ("classical" apoptosis), e.g., in thymocytes and the liver, to those dominated by autophagic degradation of cytoplasm, e.g., in the mammary gland. The induction of gene expression in these diverse types of cell death is anticipated to be different. Here we review the data regarding the regulation of apoptosis in the liver by liver tumor promoter, transforming growth factor beta-1 and related peptides as well as nutrition. In the course of hepatocarcinogenesis, initiated cells as well as preneoplastic and neoplastic cell populations showed enhanced cell replication, but also enhanced apoptosis. Tumor promoter shift the balance between birth and death by increasing the rate of cell replication and by decreasing the rate of apoptosis. Thereby, liver tumor formation is accelerated. Food restriction exhibits the opposite effect and consequently, provides protection from carcinogenesis.

Expression of myc, fos, and Ha-ras in the livers of furan-treated F344 rats and B6C3F1 mice.

Furan administered by gavage for 2 yr has been reported to induce hepatocellular carcinomas in male and female B6C3F1 mice and in male but not female F344 rats. Chronic exposure studies in our laboratory using bioassay conditions showed extensive hepatocellular toxicity and sustained increases in regenerative cell proliferation after 1, 3, and 6 wk of treatment in male and female rats and male mice. Altered expression of growth-control genes associated with this hyperproliferative state may enhance the susceptibility of these genes to mutation or may provide a selective growth advantage to preneoplastic cells. Quantitative northern blot analysis of mRNA was used to examine the expression of the oncogenes myc, fos, and Ha-ras in the livers of animals treated with furan. In male rats, a single administration of 30 mg/kg furan produced necrosis and a subsequent wave of cell proliferation 48 h after treatment and induced transient peaks in the expression of myc, fos, and Ha-ras 6-24 h after treatment. In male rat liver from our cell proliferation studies, only a slight increase in myc expression was seen at the end of week 1 of treatment. However, beginning at week 3 and increasing at week 6, up to a 15-fold increase over control values was observed in the expression of myc in the treated animals. The only other notable increase in expression observed in any animals from the cell proliferation study was a threefold increase in myc at week 6 in treated female rats. The absence of an increase in Ha-ras expression in the male mouse liver suggests that the unique pattern of Ha-ras mutations previously reported in furan-induced mouse liver tumors is not due to increased mutational susceptibility related to overexpression of this gene. The lack of sustained expression of myc, fos, and Ha-ras in rapidly proliferating liver suggests that continuous expression of these genes is not necessary to maintain increased rates of cell replication. The large increase in myc expression in male but not female rats suggests an adaptive change that may be related to the sex-specific incidence of furan-induced hepatocellular carcinomas in rats.