C-ras expression decreases during in vitro senescence in human fibroblasts

Expression of the c-ras oncogene was determined in growing early and late passage human (IMR-90) fibroblasts using northern blot analysis of total cellular RNA. It was found that late passage cells demonstrated lower levels of c-ras by about four fold when compared to levels found in early passage cells. Southern blot analysis of genomic DNA from early and late passage fibroblasts digested with either SacI or BamHI showed somewhat increased hybridization levels in early passage cells compared to late passage cells. Data is discussed in relation to a previous report of c-ras expression and gene amplification.     

Growth,morphologic, and invasive characteristics of early and late passages of a human endometrial carcinoma cell line (RL95-2)

Summary Two in vitro passages of a human endometrial adenocarcinoma continuous cell line (RL95-2), an early (subcultured <30 times) and a late passage (subcultured >200 times) have provided an interesting model to study the growth, morphologic, and invasive properties of endometrial tumors. The early passage, which has been shown to be estrogen-receptor positive, has characteristics closely resembling a primary tumor, whereas the estrogen receptor negative late passage exhibits several features of the metastatic phenotype. Compared to the early passage cells, the late passage cells were less serum dependent, formed foci, demonstrated a faster rate of growth (due to their shorter doubling times), and attained higher saturation densities. The late passage cells also displayed an altered morphology which was accompanied by alterations in the distribution of F-actin. Even though early and late passages showed similar invasive potential in an in vitro invasion assay, the late passage cells, by virtue of their several transformed characteristics, maintain distinctive properties compared with their early passage counterparts.     

Induction of DNA polymerase α in senescent cultures of normal and Werner's syndrome cultured skin fibroblasts

DNA polymerase α activity was determined following serum stimulation of early and late passages of human diploid fibroblast-like (HDFL) cultures derived from apparently normal donors (two strains) and from a patient with Werner's syndrome (one strain). Induction of this enzyme was observed in both low passage, actively proliferating cultures and in postmitotic “senescent” cultures from all three strains. The maximal polymerase activity of early and late passage cells of each strain were nearly identical when normalized to the number of cells present. However, the activity of the enzyme was observed to be significantly lower in late passage cultures when normalized to total protein content apparently because of enlargement of the senescent cells. The behavior of Werner derived cells was similar to that of the normal cells. The induction of DNA polymerase α in senescent cultures indicates that they retain the capacity to carry out some complex metabolic responses to mitogen stimulation. In addition, these results suggest the possibility that dilution of DNA polymerase α and/or other DNA replication factors may play a role in the onset or maintenance of the postmitotic state in the enlarged senescent HDFL cells.     

Heat-Induced Proteasomic Degradation of HSF1 in Serum-Starved Human Fibroblasts Aging in Vitro

The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.     

Altered regulation of c-myc expression in adenovirus-transformed cells

Expression of the oncogenes c-myc, c-raski, and p53 is studied in normal primary mouse cultures and in two adenovirus-transformed mouse cell lines. In all cases oncogene expression is measured in cells arrested in G1 (or G0 for primary cells) by serum starvation and at different times after cell cycle traverse is stimulated by addition of high serum. For primary mouse cells, c-myc mRNA levels are found to increase four- to six-fold within 1 h of serum addition and then decline by 4 h to nearly the level observed in serum-starved cells. This level is maintained throughout the remainder of the cell cycle. The early induction of c-myc is dependent on serum concentration and is independent of cell density. These results confirm and extend previous observations for primary cells. By contrast, expression of c-raski does not vary at all through the cell cycle and p53 increases with time after mitogenic stimulation. In the adenovirus-transformed cell lines, the regulation of expression of c-myc with respect to the cell cycle is altered. There is an increase in c-myc in S phase cells which is dependent on cell density and the early induction in response to serum addition as seen in primary cells is absent. Expressions of c-raski and p53 are found to show similar profiles to those observed for primary cells.     

Cyclic AMP and c-myc gene expression in PY815 mouse mastocytoma cells

The possibility was examined that inhibition of growth of PY815 mouse mastocytoma cells by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (DB cyclic AMP) results from inhibition of c-myc gene expression. Temporary increases in c-myc RNA which occurred soon after DB cyclic AMP treatment and upon removal of the drug were not consistent with direct inhibition of c-myc gene expression by DB cyclic AMP. The increases in c-myc RNA coincided with the passage through, or accumulation of cells in late G1-early S phase. It is proposed that cyclic AMP may stimulate c-myc gene expression which normally occurs only in late G1-early S phase in PY815 cells and that cyclic AMP prevents c-myc expression in cells at other phases of the cell cycle by inhibiting their progression past a cyclic AMP-sensitive restriction point in early G1 phase.     

Glycosaminoglycan production in cultures of early and late passage human endothelial cells: the influence of an anionic endothelial cell growth factor and the extracellular matrix

An endothelial cell (EC) growth factor isolated from bovine brain stimulates in vitro growth of human umbilical vein endothelial cells, and permits long term serial propagation. In the presence of increasing concentrations of EC growth factor, confluent cultures of early (CPDL less than or equal to 20) and late (CPDL greater than 20) passage human endothelial cells exhibit an increased incorporation of 3H-glucosamine and Na235SO4 into the glycosaminoglycans (GAG), hyaluronic acid, chondroitin, chondroitin-4-sulfate, dermatan-4-sulfate, and chondroitin-6-sulfate. An increase in both labelled sulfated and nonsulfated GAG was observed in the cytosol, membrane, secreted and extracellular matrix fractions. In contrast, endothelial cells grown in the presence of EC growth factor contained decreased amounts of labelled heparan sulfate than cells grown without EC growth factor. Confluent cultures of early passage cells had significantly more labelled GAG but significantly less heparan sulfate than cultures of late passage cells on a per cell basis. Extracellular matrix from early passage cells contained about two- to seven-fold more labelled GAG than extracellular matrix from late passage cells, but only about half as much labelled heparan sulfate. Cell adhesion was enhanced when cells were grown in the presence of EC growth factor as compared to adhesion of cells grown without EC growth factor. Conversely, trypsin-mediated detachment of cells grown in the presence of growth factor was inhibited as compared to detachment of cells grown in medium without EC growth factor. The composition of the extracellular matrix influenced incorporation of labelled GAG into extracellular matrix. Early passage cells grown to confluence on a matrix from late passage cells incorporated significantly less labelled GAG into extracellular matrix than when grown to confluence on matrix from early passage cells. Incorporation of labelled GAG into extracellular matrix was significantly higher when late passage cells were grown on a matrix from early passage endothelial cells than when grown on matrix from late passage cells. We conclude that EC growth factor selectively stimulates incorporation of isotopic precursors into GAG in cultures of early and late passage endothelial cells but inhibits incorporation of radiolabel into heparan sulfate; early passage cells contain more GAG but less heparan sulfate than late passage cells, extracellular matrix controls the amount of GAG and heparan sulfate incorporated into matrix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Messenger RNAs coding for enzymes of polyamine biosynthesis are induced during the G0-G1 transition but not during traverse of the normal G1 phase

The events occurring during emergence of cells from quiescence ("G0") are not necessarily identical to those in the G1 phase of continuously dividing cells. Cellular levels of the mRNAs coding for ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SDC), key enzymes in polyamine synthesis, increased maximally within 5 h after addition of serum to resting 3T3 cells, following a kinetic course similar to that of c-myc mRNA. In a pure early G1 population of cells, prepared by centrifugal elutriation of growing fibroblasts, the levels of ODC and SDC mRNAs were not significantly lower than in other phases of the cell cycle and approximated serum-induced levels rather than the reduced values found in serum-starved cells. Thus, we conclude that the mRNAs coding for the polyamine biosynthetic enzymes, like c-myc, are growth controlled, but not regulated during traverse of a normal cell cycle.     

Enhanced synthesis of a Mr = 55,000 dalton peptide by senescent human fibroblasts

The secretory protein profiles of early and late passage cultures of human fibroblasts were compared using polyacrylamide gel electrophoresis. In comparison with early passage cell cultures (40-50% lifespan completed), late passage (greater than 80% of lifespan completed) cell cultures exhibited enhanced production of several peptides in the Mr range 55-60,000. One of those peptides had an apparent molecular weight of Mr = 55,000 and was constitutively present in the late passage cell conditioned medium. Late passage cell cultures synthesized the Mr = 55,000 peptide in the presence or absence of fetal bovine serum. Serum did not enhance its production by early passage cells. Further, production of the peptide was not induced in early passage cell cultures whose proliferation was arrested either by serum starvation or by contact inhibition. Pulse chase studies demonstrated that the peptide appears in the culture medium within 60 min of labeling. There was no evidence that it is derived via degradation of other proteins present either in early passage or late passage cell conditioned media. Further, the production of the 55,000 dalton peptide did not appear to be regulated by factors present in conditioned media. The peptide was detected in the conditioned media produced by late passage cultures of several different cell strains.