Novel mechanisms of sublethal oxidant toxicity: induction of premature senescence in human fibroblasts confers tumor promoter activity

Aging is the highest risk factor for cancer. Although oxidants are thought to contribute to both aging and cancer, the interplay between oxidative stress, aging, and cancer has not been well studied. Human diploid fibroblasts (HDFs) undergo premature senescence in response to sublethal doses of H(2)O(2). To test the hypothesis that senescent or senescent-like HDFs function as a tumor promoter, we have employed an in vitro skin tumor promotion model, in which colony formation is measured using initiated mouse keratinocyte 308 cells seeded at clonal density. 308 cells form colonies when co-cultured with normal HDFs only in the presence of the tumor promoter phorbol 12-myristate 13-acetate (TPA), which induces an average of 5.75 colonies. When co-cultured with H(2)O(2)-treated HDFs, 308 cells form an average of 30.3 colonies. To understand the mechanism behind this phenomenon, we tested whether conditioned medium of HDFs, HDF extracellular matrix (ECM), density of HDFs, or the contact between keratinocytes and HDFs plays a role in 308 cell colony formation. The conditioned medium from prematurely senescent cells resulted in an average of eightfold more 308 cell colonies formed than the conditioned medium from normal HDFs, and the growth-promoting effect of the conditioned medium was trypsin sensitive. The ECM alone was not able to induce 308 cell colony formation. Increasing the density of normal HDFs or contact with normal HDFs but not senescent-like HDFs was inhibitory to the growth of 308 cells. Measurement of Connexin 43 indicated a decreased expression of the protein, which suggests an impaired gap junction communication in senescent-like HDFs. We conclude that H(2)O(2)-treated fibroblasts not only lose contact inhibition of the growth of initiated keratinocytes perhaps related to reduced gap junction communication but also increase production of secreted protein factors to enhance the growth of 308 keratinocytes.     

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.     

Insulin-like growth factor-binding protein-1 inhibits binding of IGF-I on fetal skin fibroblasts but stimulates their DNA synthesis

Insulin-like growth factor-binding protein-1 (IGFBP-1) was purified from human midtrimester amniotic fluid using monoclonal anti-IGFBP-1 affinity column. Two peaks were obtained in anion exchange chromatography. Both had the same molecular mass of 30 kDa. In monolayer cultures of fetal skin fibroblasts both forms of IGFBP-1 inhibited binding of [125I]IGF-I onto the cells, but amplified the IGF-I-stimulated [3H]thymidine incorporation into the same cells. Radiolabeled IGFBP-1 did not bind to the cells. No detectable IGFBP-1 was released into conditioned medium from the cells, and they contained no specific IGFBP-1 mRNA. Recently we found that the same IGFBP-1 preparation inhibits IGF-I-stimulated [3H]thymidine incorporation into human hyperstimulated granulosa cells. These results show that, depending on target cells, the same protein is capable of either stimulating or inhibiting DNA synthesis.     

Subcytotoxic H2O2 stress triggers a release of transforming growth factor-beta 1, which induces biomarkers of cellular senescence of human diploid fibroblasts

Stress-induced premature senescence (SIPS) is induced 3 days after exposure of human diploid fibroblasts to subcytotoxic oxidative stress with H(2)O(2), with appearance of several biomarkers of replicative senescence. In this work, we show that transforming growth factor-beta1 (TGF-beta1) regulates the induction of several of these biomarkers in SIPS: cellular morphology, senescence-associated beta-galactosidase activity, increase in the steady-state level of fibronectin, apolipoprotein J, osteonectin, and SM22 mRNA. Indeed, the neutralization of TGF-beta1 or its receptor (TGF-beta RII) using specific antibodies decreases sharply the percentage of cells positive for the senescent-associated beta-galactosidase activity and displaying a senescent morphology. In the presence of each of these antibodies, the steady-state level of fibronectin, osteonectin, apolipoprotein J, and SM22 mRNA is no more increased at 72 h after stress. Results obtained on fibroblasts retrovirally transfected with the human papillomavirus E7 cDNA suggest that retinoblastoma protein (Rb) regulates the expression of TGF-beta1 in stressful conditions, leading to SIPS and overexpression of these four genes.     

Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated beta-galactosidase (SA-beta-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-beta-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest.     

Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.     

Bcl-2 protects against oxidative stress while inducing premature senescence

Replicative senescence is a cellular response to stress that has been postulated to serve as a tumor suppression mechanism and a contributor to aging. Numerous experimental studies have demonstrated that an apparently identical senescent state can also be prematurely induced in vitro in different cell types following sublethal oxidative stress stimuli. The former suggests a molecular link between cell cycle regulation and cell survival that could involve regulatory proteins such as Bcl-2. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. The aim of this work was to determine if the protection against oxidative stress mediated by Bcl-2 could prevent or delay oxidative stress-induced senescence. Using a retroviral infection system, Bcl-2 was overexpressed in primary, nonembryonic mice fibroblasts obtained from lungs derived from 2-month-old CD1 mice. Fibroblasts overexpressing Bcl-2 were exposed to 75 microM H2O2 for 2 h to induce SIPS. The rate of proliferation and the increment of senescent cells were then determined. Our results indicate that overexpression of Bcl-2 protected primary fibroblasts against oxidative stress-mediated reduction in cell proliferation, but did not prevent premature senescence.     

Intact insulin-like growth factor binding protein-5 (IGFBP-5) associates with bone matrix and the soluble fragments of IGFBP-5 accumulated in culture medium of neonatal mouse calvariae by parathyroid hormone and prostaglandin E2-treatment

We examined the distribution of insulin-like growth factor binding proteins (IGFBPs) in cultured neonatal mouse calvariae. IGFBP-3 and -4 were predominantly found in the conditioned medium. IGFBP-2 was partitioned between conditioned medium and bone and extracellular matrix (BECM), while intact (31-kDa) IGFBP-5 was most abundant in BECM extracts. After treatment with parathyroid hormone (PTH, 10⁻⁸ M) or prostaglandin E₂ (PGE₂, 10⁻⁶ M), immunoreactive IGFBP-5 accumulated in the conditioned medium in a 21-kDa form which did not bind IGF-I on Western ligand blots. PTH and PGE₂ did not alter the level of steady-state IGFBP-5 mRNA, nor markedly stimulate IGFBP-5 synthesis in the calvariae, and thus accumulation of 21-kDa IGFBP-5 was largely due to release from BECM. This accumulation of truncated IGFBP-5 in the conditioned medium was not dependent on osteoclastic bone resorption, since it was not blocked by calcitonin or a bisphosphonate which inhibited PTH- and PGE₂-stimulated ⁴⁵Ca-release. The conditioned medium from PTH- or PGE₂-treated cultures degraded recombinant human IGFBP-5 into lower molecular weight fragments. Addition of IGF-I at 10⁻⁸ M into the culture resulted in accumulation of native 31-kDa IGFBP-5. However, even in the presence of IGF-I, the native IGFBP-5 was degraded and the 21-kDa product accumulated in the culture medium. These results suggested a possible proteolytic mechanism for 21-kDa IGFBP-5 accumulation, responsive to PTH and PGE₂. Aprotinin, leupeptin, cystatin, and bestatin did not inhibit the effects of PTH and PGE₂ in the cultures. The localization of IGFBP-5 in BECM and its release and proteolysis induced by PTH and PGE₂ could play a role in the local regulation of bone metabolism. © 1996 Wiley-Liss, Inc.     

Inhibition of Caco-2 cell proliferation by all-trans retinoic acid: role of insulin-like growth factor binding protein-6.

The present study examined the effects of all-trans retinoic acid (tRA) on proliferation and expression of the IGF system in Caco-2 human colon adenocarcinoma cells. tRA inhibited Caco-2 cell proliferation in a dose-dependent manner, with a 40 +/- 2% decrease in cell number observed 48 h after the addition of 1 microM tRA. Ligand blot analysis of IGFBPs in conditioned media revealed that Caco-2 cells produced three IGFBPs of M(r): 34,000 (IGFBP-2), 24,000 (IGFBP-4), and 32,000 (IGFBP-6). The concentrations of IGFBP-2 and IGFBP-4 decreased by 48 +/- 6 and 70 +/- 13%, respectively, whereas that of IGFBP-6 increased by 698 +/- 20% with 1 microM tRA. tRA decreased mRNA levels of IGFBP-2 and IGFBP-4 by 20 +/- 3 and 50 +/- 8%, respectively, whereas tRA increased IGFBP-6 mRNA by 660 +/- 20%. tRA did not alter levels of IGF-II mRNA or peptide. To examine if endogenous IGFBP-6 inhibits cell proliferation, Caco-2 cells were transfected with an IGFBP-6 cDNA expression construct or pcDNA3 vector only and stable clones were selected. Clones overexpressing IGFBP-6 grew more slowly than vector controls and achieved final densities 30-55% lower than those of vector controls. Accumulation of IGFBP-6 mRNA and concentrations of IGFBP-6 peptide in conditioned media were increased by 200-250 and 220-250%, respectively, in the IGFBP-6 clones compared with controls. Increased expression of IGFBP-6, which has a high binding affinity for IGF-II, following tRA treatment suggests that the decreased proliferation caused by tRA may result, at least in part, from IGFBP-6-mediated disruption of the IGF-II autocrine loop in these colon cancer cells.     

Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells

Platelet-derived growth factor (PDGF) is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We investigate whether PDGF plays a role in the regulation of the adapter protein Disabled-2 (DAB2) that expresses abundantly in platelets and megakaryocytes. Western blot analysis revealed that conditioned medium from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated, megakaryocytic differentiating K562 cells upregulated DAB2 expression. DAB2 induction and megakaryocytic differentiation was abrogated when cells were co-treated with the PDGF receptor inhibitor STI571 or when the conditioned medium was derived from TPA-plus STI571-treated cells. Although the level of PDGF mRNA was not altered by STI571, an approximate 44% decrease in PDGF in the conditioned medium was observed. Consistent with these findings, interfering PDGF signaling by PDGF neutralization antibody or dominant negative PDGF receptors attenuated DAB2 expression. Accordingly, transfection of an expression plasmid encoding secreted PDGF upregulated DAB2. This study shows for the first time that PDGF autocrine signaling regulates DAB2 expression during megakaryocytic differentiation.     

HIV-infected lymphocytes regulate fibronectin synthesis by TGF beta 1 secretion

Alterations in lymph node architecture occur with HIV infection and contribute to immunological derangements. We previously showed that matrix fibronectin stabilized HIV and increased HIV infection of PBL. We showed increased fibronectin deposition in lymph nodes of HIV-infected patients. However, we did not detect a difference in fibronectin synthesis between uninfected and infected PBL. Therefore, we hypothesized that interactions of HIV-infected cells with fibroblasts resulted in increased fibronectin deposition. We detected increased fibronectin deposition by immunofluorescence on fibroblasts cocultured with HIV-infected PBL. We also found a 6-fold increase in fibronectin mRNA levels in fibroblasts cocultured with HIV-infected PBL by real-time PCR. Furthermore, when HIV-infected PBL were added to reporter fibroblasts stably transfected with a fibronectin promoter, we found a 1.5- to 2-fold increase in promoter activity. Since conditioned medium from HIV-infected PBL also increased fibronectin promoter activity, we hypothesized that a soluble factor such as TGFbeta was responsible for increased fibronectin secretion. Pretreatment of supernatant from HIV-infected PBL with a neutralizing Ab to TGFbeta1 abrogated the increased fibronectin promoter activity. We confirmed that HIV-infected PBL produced increased TGFbeta1 by ELISA. Using Mv1Lu reporter cells, we found a 2- to 3-fold increase in biologically active TGFbeta in supernatants of HIV-infected PBL. Finally, we determined that HIV infection did not change the percentage of active TGFbeta. Our data suggest that HIV-infected lymphocytes indirectly contribute to lymph node remodeling by secretion of TGFbeta1, which increases fibronectin synthesis by fibroblasts.     

Mechanical strain increases type I collagen expression in pulmonary fibroblasts in vitro.

Tissue remodeling is an adaptive response to mechanical tension in the lung. However, the role of pulmonary fibroblasts in this response has not been well characterized. This study investigates the influence of extracellular matrix on the response of fibroblasts to mechanical strain. Cells were cultured on flexible-bottom surfaces coated with fibronectin, laminin, or elastin and exposed to strain. Under these conditions, fibroblasts align perpendicular to the force vector. This stimulus results in an increase in alpha(1)(I) procollagen mRNA in cells cultured on laminin or elastin but not fibronectin. Increased alpha(1)(I) procollagen mRNA was detected 6 h after exposure to strain and reached control levels by 72 h. [(3)H]proline incorporation into newly synthesized procollagen reflects changes in mRNA levels. Strained fibroblasts cultured on laminin or elastin incorporated 190 and 114%, respectively, more [(3)H]proline into procollagen than did unstrained cells. No difference was detected in strained fibroblasts cultured on fibronectin. These results suggest that fibroblasts respond to mechanical strain in vitro, and this response is signaled by cell-extracellular matrix interactions.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.