Density-inhibited Cells Research Articles

High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing the lowest PiT1 mRNA levels. The mechanism behind the role of PiT1 in increased cell proliferation is not known. We, however, found that compared to control cells, cultures of NIH3T3 cells overexpressing PiT1 upon seeding showed increased cell number after 24h and had shifted more cells from G0/G1 to S+G2/M within 12h, suggesting that an early event may play a role. We here show that expression of human PiT1 in NIH3T3 cells led to faster cell adhesion; this effect was not cell type specific in that it was also observed when expressing human PiT1 in MC3T3-E1 cells. We also show for NIH3T3 that PiT1 overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby contribute to their proliferative advantage within the first 24h after seeding.

Regulation of cell proliferation and cell density by the inorganic phosphate transporter PiT1

AbstactBackgroundThe inorganic phosphate (Pi) transporter, PiT1 (SLC20A1), is ubiquitously expressed in mammalian cells. It has previously been shown that down-regulation of PiT1 severely impaired the proliferation of two transformed human cells lines, HepG2 and HeLa, and the tumorigenicity of HeLa cells in nude mice. Moreover, PiT1 knock-out mice do not survive past E12.5 and from E10.5, the embryos were found to be growth-retarded and showed reduced proliferation of liver cells. Isolated mouse embryonic fibroblasts with knocked out as well as reduced PiT1 expression levels also exhibited impaired proliferation. Together these results suggest that a certain level of PiT1 is important for proliferation. We have here investigated the role of PiT1 in regulation of cell proliferation using two strictly density-inhibited cells lines, the murine MC3T3-E1 and NIH3T3 cells.ResultsWe found that knock-down of PiT1 in MC3T3-E1 cells led to impaired proliferation supporting that at least a certain level of PiT1 is important for wildtype level of proliferation. We, however, also observed that MC3T3-E1 and NIH3T3 cells themselves regulate their endogenous PiT1 mRNA levels with lower levels in general correlating with decreased proliferation/increased cell density. Moreover, over-expression of human PiT1 led to increased proliferation of both MC3T3-E1 and NIH3T3 cultures and resulted in higher cell densities in cultures of these two strictly density-inhibited cell lines. In addition, when we transformed NIH3T3 cells by cultivation in fetal bovine serum, cells over-expressing human PiT1 formed more colonies in soft agar than control cells.ConclusionsWe conclude that not only is a certain level of PiT1 necessary for normal cell division as suggested by previously published studies, rather the cellular PiT1 level is involved in regulating cell proliferation and cell density and an increased PiT1 expression can indeed make NIH3T3 cells more sensitive to transformation. We have thus provided the first evidence for that expression of the type III Pi transporter, PiT1, above the endogenous level can drive cell proliferation and overrule cell density constraints, and the results bridge previous observations showing that a certain PiT1 level is important for regulating normal embryonic growth/development and for tumorigenicity of HeLa cells.

Restoration of Functional Gap Junctions through Internal Ribosome Entry Site-Dependent Synthesis of Endogenous Connexins in Density-Inhibited Cancer Cells

Gap junctions are composed of connexins and are critical for the maintenance of the differentiated state. Consistently, connexin expression is impaired in most cancer cells, and forced expression of connexins following cDNA transfection reverses the tumor phenotype. We have found that the restoration of density inhibition of human pancreatic cancer cells by the antiproliferative somatostatin receptor 2 (sst2) is due to overexpression of endogenous connexins Cx26 and Cx43 and consequent formation of functional gap junctions. Immunoblotting along with protein metabolic labeling and mRNA monitoring revealed that connexin expression is enhanced at the level of translation but is not sensitive to the inhibition of cap-dependent translation initiation. Furthermore, we identified a new internal ribosome entry site (IRES) in the Cx26 mRNA. The activity of Cx26 IRES and that of the previously described Cx43 IRES are enhanced in density-inhibited cells. These data indicate that the restoration of functional gap junctions is likely a critical event in the antiproliferative action of the sst2 receptor. We further suggest that the existence of IRESes in connexin mRNAs permits connexin expression in density-inhibited or differentiated cells, where cap-dependent translation is generally reduced.

The role of protein tyrosine phosphatases in density-dependent growth control of normal rat kidney cells

In normal rat kidney cells protein tyrosine phosphatases (PTPases) play a role in attaining density-dependent growth arrest after stimulation with mitogens. The PTPase inhibitor sodium orthovanadate prevents density-dependent growth inhibition of EGF-treated cells and mimicks in that respect the action of TGFβ and retinoic acid. However, enhanced PTPase activity is not obligatory for maintaining cells in a density-arrested state. In contrast to TGFβ and retinoic acid, vanadate is unable to restimulate density-inhibited cells, indicating that different mechanisms are operating. Yet, vanadate is strongly potentiating the effect of low concentrations of TGFβ but not of retinoic acid, implicating that tyrosine phosphorylation is linked to TGFβ action and that PTPase may represent a negative control element in the TGFβ signaling pathway.

The short-term increase in extracellular potassium concentration causes DNA replication onset in density-inhibited cells

The high potassium concentration effect on the human diploid fibroblasts (HDF) and 3T3 cells was investigated. The incubation of confluent cultures of HDF or 3T3 Swiss cells in the medium with 50 m M K + for 35 min induced, 12 h later, the onset of DNA replication in a significant proportion of culture cell population. The same treatment had no effect upon the sparce cell cultures. No stimulation of DNA replication was observed in the absence of serum in culture medium.

Killing and mutation induction in quiescent V79 cells as influenced by nalidixic acid

Involvement of topoisomerase II in the repair of damage by N-methyl- N′-nitro- N-nitrosoguanidine, UV light and γ-rays has been studied using quiescent V79 cells. In each case, the presence of nalidixic acid, the inhibitor of topoisomerase II, decreased the survival through suppression of potentially lethal damage repair. There was also an increase in the yield of mutants because of such suppression. The observations were in contrast with those made with exponential cells in the following aspects: (a) density-inhibited cells showed a positive response to suppression of topoisomerase II activity after treatment with UV light and γ-rays and (b) for MNNG exposure, mutational yield increased instead of decreasing as in exponential cells. The results showed that topoisomerase II played an important part in the repair of damage of density-inhibited cells.

Loss of focal contacts accompanies: The density dependent inhibition of cell growth

The formation of focal contacts by fibroblast-like 3T3 and 9390 cells and endothelial XTH-2 cells was examined with the use of reflection interference contrast microscopy at different cell densities. It was found that cells density-inhibited in growth loose all focal contacts with the substratum. This observation explains the disappearance of stress fibers in density-inhibited cells, and also offers an explanation for the mechanism of density-dependent inhibition of proliferation of anchorage-dependent cells.

Effects of epidermal growth factor and platelet-derived growth factor on c- fos and c- myc mRNA levels in normal human fibroblasts

The mRNA levels of two proto-oncogenes, c- fos and c- myc, were determined in human foreskin fibroblasts exposed to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) in a serum-free, defined medium (MCDB 104). Untreated, quiescent cells were found to have low or undetectable levels of c- fos and c- myc mRNA. Within 10 min after the addition of EGF or PDGF the c- fos mRNA level increased, reached a peak at 30 min, and then declined to the control level after 60 min. The level of c- myc mRNA increased somewhat later and peaked after 8 h in cultures treated with either of the growth factors. The c- myc mRNA level remained elevated throughout the 24 h of investigation. The concentrations of EGF and PDGF required for a maximal effect on c- fos or c- myc expression were found to be similar to those that give maximal effect on cell proliferation. Both c- fos and c- myc mRNA expression were superinduced by the addition of cycloheximide. The addition of neutralizing PDGF antibodies to cultures that had received PDGF 4 h earlier inhibited the subsequent increase in the c- myc mRNA level, indicating that the effect of PDGF on c- myc expression is not caused by a “hit and run” mechanism. Density-inhibited cells responded to EGF and PDGF by an increase in c- fos and c- myc mRNA levels in the absence of any mitogenic response. The present results conform to the view that the c- fos and c- myc proto-oncogenes may be important (or necessary) but not sufficient for the initiation of DNA synthesis. Moreover, the finding that both EGF and PDGF increase c- fos and c- myc expression supports our previous suggestion that these two growth factors may in part act via a common intracellular pathway in the prereplicative phase of human fibroblasts.

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low- M r chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher- M r HMG proteins (HMG 1 + 2) to the lower- M r HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, 32P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the 32P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [ 3H]adenosine showed a different pattern of label distribution; the higher- M r HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower- M r HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [ 32P]orthophosphate or [ 3H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.

Influence of Caffeine on X-Ray-Induced Killing and Mutation in V79 Cells

Effects produced by caffeine on X-irradiated Chinese hamster V79 cells depended on the growth conditions of the cells. For exponentially growing cells, nontoxic concentrations of caffeine decreased the shoulder width from the survival curve, but the slope remained unchanged. The yield of mutants under the same conditions also remained unaffected. In case of density-inhibited cells, delaying trypsinization for 24 h after X irradiation increased the survival and decreased the yield of mutants. The presence of caffeine during this incubation period inhibited such recovery and significantly increased the yield of X-ray-induced mutants.

Enhanced expression of X-ray- and UV-induced chromosome aberrations by cytosine arabinoside in ataxia telangiectasia cells

The effect of cytosine arabinoside (ara-C) on the frequency of X-ray- or UV-induced chromosome aberrations was studied in cultured skin fibroblasts derived from 2 normal persons, 4 ataxia telangiectasia (AT) patients and 2 obligate AT heterozygotes. Density-inhibited cells were irradiated with X-rays or UV, post-treated with ara-C, and chromosomes in the first port-irradiation mitoses were examined. UV, a poor induced of chromosome-type aberrations in G 1, caused chromosome-type aberrations (dicentrics and rings) when coupled with ara-C both in normal and AT cells, but to a much greater extent in AT cells. In AT cells, an elevated induction of both terminal deletions and chromatid aberrations was also observed by the application of UV and ara-C, and unexpectedly, UV alone induced a considerable frequency of both types of aberration. The enhancing effect of ara-C on X-irradiated cells was less pronounced than on UV-irradiated cells. The response of AT heterozygotes were virtually the same as those of normal cells. These findings suggest that ara-C can convert the UV-induced DNA damage into the type that has a potential to induce dicentrics and rings in G 1 as well as to elicit a hypersensitive response of AT cells.

The induction of SCE with relation to specific base methylation of DNA in Chinese hamster cells by N-methyl-N-nitrosourea and dimethylsulfate.

The relationship between the formation of alkylated purines in DNA and sister chromatid exchange (SCE) induction has been studied. Both exponentially growing and density-inhibited Chinese hamster (V79) cultures were treated with various doses of N-methyl-N-nitrosourea (MNU) or dimethylsulfate (DMS). The colony-forming ability and induced frequencies of SCEs were assayed. Following the exposure of density-inhibited cells to radiolabeled methylating agents these phenomena were related to the levels of 7-methylguanine (7-meGua), 0(6)-methylguanine (0(6)-meGua) and 3-methyladenine (3-meAde) in the DNA. At equitoxic doses MNU and DMS induced similar frequencies of SCEs. Since, at equitoxic doses MNU produces about 20 times more 0(6)-meGua in V79-cell DNA than does DMS, this indicates that the formation of this adduct in DNA is not critical for the induction of SCEs by these alkylating agents. Dimethylsulfate-induced SCEs may be mediated via the production of both 3-meAde and 7-meGua in the DNA; these 2 methylated purines may also be responsible for MNU-induced SCE. No one specific methylated purine was identified, therefore, as being solely accountable for the formation of SCEs. The repair of lesions in the DNA of nonreplicating V79 cells lead to a reduction in the SCE frequency on their subsequent release from the density-inhibited state. This suggests that excision repair is not responsible for the formation of SCEs.

Growth-state independent induction of specific proteins in Swiss 3T3 cells by interferon.

The early effects of interferon (IFN) on the pattern of protein synthesis in Swiss 3T3 mouse fibroblasts were investigated by two-dimensional gel electrophoresis of 35S-methionine labeled cell extracts. IFN induced within 4 h the synthesis of three proteins with molecular weights of 48, 49, and 50 kD. An increase in the rate of synthesis of a 31 kD protein was also found. These changes were observed after IFN treatment of exponentially growing cells, density-inhibited cells, serum-starved cells, and serum-stimulated quiescent cells. The same protein patterns were obtained from cells treated with IFN-alpha or IFN-beta. The IFN response was blocked by the addition of actinomycin D, implying de novo transcription of the corresponding species of messenger RNA.

Initiation of carcinogenesis dependence upon MNNG-induced release of the G1 block of density-inhibited Syrian hamster cells.

After N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment of density-inhibited postconfluent (DDIR) Syrian hamster embryo cell cultures, only a small population of the cells incorporate [3H]-thymidine within the next 10-20 h. The transformed colonies, observed subsequent to reseeding, are derived from the population incorporating [3H]-thymidine. This study demonstrates that MNNG-induced thymidine incorporation resulted from semiconservative DNA synthesis as analyzed by DNA density gradients. Furthermore, cell cycle distributions at selected times after treatment indicate that the MNNG-responsive population was released from the density-dependent G1 block, proceeded through S, and became blocked again at G2 or M. These results indicate that DNA synthesis is temporally related to an early step in carcinogenesis.

Cell growth regulation during density inhibition and interferon treatment

The growth of P 3HR-1, a Burkitt' s lymphoma cell line, was regulated differently at high cell numbers and upon treatment with human leukocyte interferon (IFN-α). At terminal cell titre, cell growth was reduced by progressing through the cell cycle more slowly, whereas IFN-treated cells escaped from the cell cycle by going into a G0-like state. Furthermore, in comparison with density-inhibited cells, IFN-treated cells had, upon dilution into fresh medium, a longer lag period before they began to grow exponentially again. No G0/G1 restriction point was apparent in this cell line. These cells had to undergo one mitotic division before any IFN-induced inhibition of proliferation could be observed.

The induction of SCE and chromosomal aberrations with relation to specific base methylation of DNA in Chinese hamster cells by N-methyl-N-nitrosourea and dimethyl sulphate.

Chinese hamster cells (V79) were treated, either as exponentially proliferating cultures or under conditions where they were density-inhibited, with various doses of the potent carcinogen N-methyl-N-nitrosourea (MNU) or the relatively weak carcinogen dimethylsulphate (DMS). The colony forming ability of these cells and the induced frequencies of sister chromatid exchanges (SCEs) and chromosomal aberrations were assayed. Following the exposure of density-inhibited cells to radio-labelled methylating agents (labelled in the methyl group) these phenomena were related to the levels of 7-methylguanine (7-meGua), O6-methylguanine (O6-meGua) and 3-methyladenine (3-me-Ade) in the DNA. At equitoxic doses MNU and DMS induced similar frequencies of SCEs and chromosomal aberrations. Since, at equitoxic doses, MNU produces approximately 20 times more O6-meGua in V79 cell DNA than does DMS, this indicates that the formation of O6-meGua in DNA is not a major cause of SCEs and chromosomal aberrations. DMS-induced SCEs may be mediated via the production of both 3-meAde and 7-meGua in the DNA; these two methylated purines may also be responsible for MNU-induced SCEs. Therefore, no one specific methylated purine was identified as being solely accountable for the formation of SCEs. Also, the repair of lesions in the DNA of non-replicating V79 cells leads to a reduction in the SCE frequency on their subsequent release from the density-inhibited state, suggesting that repair is not intimately responsible for their formation. No association was discernable between chromosomal aberrations and any of the three methylated purines studied.

The effect of phorbol esters on the proliferation of C3H-10T 1/2 mouse fibroblasts: consideration of both stimulatory and inhibitory effects.

TPA stimulates cell cycle activation in both serum-deprived and density-inhibited cultures. The cells reestablish cycle arrest after no more than one generation, and addition of fresh drug produces no further response. However, cells freshly trypsinized can respond with a series of repetitive generations resulting in 3.5-4.0 population doublings over 72 hrs. In kinetic pulse experiments TPA enhanced 3H-thymidine incorporation in density-inhibited cells stimulated by fresh serum but only after markedly suppressing incorporation 8-13 hrs after serum stimulation. When cells arrested by serum deprivation were pretreated with TPA, fresh serum stimulation led to initiation of 3H-TdR incorporation 5 hrs earlier than untreated controls. However, TPA addition at the time of serum stimulation did not lead to a suppression at 8-13 hrs, whereas enhancement was observed during peak incorporation times regardless of whether the cells were pretreated with TPA during serum deprivation. The results support the concept that there can exist within G1 multiple states of responsiveness to phorbol esters. These pharmacologically induced states may be correlated with corresponding physiological states of the G1 phase of cell cycle.

Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle.

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum-stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.

Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma-derived cell lines.

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40-transformed teratocarcinoma-derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density-inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.

Potassium fluxes and ouabain binding in growing, density‐inhibited and rous sarcoma virus‐transformed chicken embryo cells

Potassium fluxes, ouabain binding, and Na+ and K+ intracellular concentrations were determined for cultures of growing normal, density-inhibited and Rous sarcoma virus-transformed chicken embryo fibroblasts. No significant differences in K+ influx or ouabain binding were detected between growing normal cells and Rous sarcoma virus-transformed cells; however, ouabain binding and ouabain-sensitive K+ influx were 1.5- to 1.8-fold lower in density-inhibited cells. Thus, potassium influx in this system can be classified as a growth-related, but not transformation-specific change. As determined by both flame photometry and radioisotopic (42K) equilibration, growing normal and density-inhibited cells had similar potassium contents, whereas transformed cells exhibited 1.4-fold higher potassium levels. Sodium ion levels, as measured by flame photometry, were also 2- to 4.5-fold higher in transformed than normal or density-inhibited cells. Complementary studies of potassium efflux showed a 1.3- to 1.5-fold higher rate (based on the percentage of pool exiting the cell) in growing normal versus density-inhibited or transformed fibroblasts. Because of the larger potassium pool in transformed cells, efflux based on absolute number of potassium ions is similar in normal and transformed chicken embryo fibroblasts.