Expression of p24, a novel p21Waf1/Cip1/Sdi1-related protein, correlates with measurement of the finite proliferative potential of rodent embryo fibroblasts

Normal mammalian fibroblasts undergo a limited number of divisions when cultured in vitro before entering a state of replicative senescence. The molecular basis for the determination of the finite mitotic potential is not known. Nevertheless, simian virus 40 T antigen, among other oncogenes, is able to prevent senescence in rodent embryo fibroblasts. T antigen immortalized cells are dependent upon this protein for maintaining growth once their normal mitotic life span has elapsed. Even though the mechanism that measures the finite mitotic potential of rodent fibroblasts is not known, it has been shown that it continues to function normally in the presence of this immortalizing gene. Accumulation of cyclin-dependent kinase inhibitors such as p21^{Waf1/Cip1/Sdi1} could potentially be a component of the mechanism that determines the finite life span. Here we show that accumulation of p21^{Waf1/Cip1/Sdi1} does not correlate with this biological counting mechanism, but we have identified p24, a p21^{Waf1/Cip1/Sdi1}-related protein, whose accumulation does correlate with the measurement of the finite proliferative potential of rodent embryo fibroblasts and suggest that sequestration might be a mechanism by which its activity is regulated.     

Surveillance mechanism linking Bub1 loss to the p53 pathway

Bub1 is a kinase believed to function primarily in the mitotic spindle checkpoint. Mutation or aberrant Bub1 expression is associated with chromosomal instability, aneuploidy, and human cancer. We now find that targeting Bub1 by RNAi or simian virus 40 (SV40) large T antigen in normal human diploid fibroblasts results in premature senescence. Interestingly, cells undergoing replicative senescence were also low in Bub1 expression, although ectopic Bub1 expression in presenescent cells was insufficient to extend lifespan. Premature senescence caused by lower Bub1 levels depends on p53. Senescence induction was blocked by dominant negative p53 expression or depletion of p21(CIP1), a p53 target. Importantly, cells with lower Bub1 levels and inactivated p53 became highly aneuploid. Taken together, our data highlight a role for p53 in monitoring Bub1 function, which may be part of a more general spindle checkpoint surveillance mechanism. Our data support the hypothesis that Bub1 compromise triggers p53-dependent senescence, which limits the production of aneuploid and potentially cancerous cells.     

Replicative senescence and differentiated gene expression in cultured adrenocortical cells

We have used a differentiated endocrine cell type, the adrenocortical cell, to investigate the interrelationship of senescence and differentiation, the effects of the environment on differentiated gene expression, and the interrelationship of differentiated gene expression and proliferation. In bovine adrenocortical cells, expression of some differentiated functions is maintained to very late points in the replicative life span, whereas expression of others is lost at various times prior to senescence. There isclonal variation in the rate and extent of loss of functions. For steroid 17-hydroxylase, in situ hybridization shows that the observed decline in induction of 17-hydroxylase mRNA during senescence results from a decline in the fraction of cells expressing the gene. Descendants of expressing cells in the primary cell population randomly become nonexpressing. Among clones there is a correlation between the fraction of cells expressing the gene and remaining replicative potential, although several experiments show no direct mechanism linking replicative senescence and 17-hydroxylase expression. Transfection with SV40 early region genes also dissociates the decline in growth and the change in 17-hydroxylase expression. SV40 T antigen selectively affects growth; expression of 17-hydroxylase is stabilized either in the on state, when cells are transfected early in the culture life span, or in the off state, when senescent cells are transfected. Thus, although the switching off of 17-hydroxylase expression and the loss of replicative potential are independent events, the switching process requires DNA replication. Because the switch is irreversible, changes in replicative potential occurring after the switch-off event do not affect the state of expression of the switched-off gene. Changes in differentiated cell properties and the changes in replicative potential may be two facets of a general phenomenon of stochastic changes in gene expression in normal cells during senescence.     

A model for the phenotypic presentation of Werner's syndrome.

Werner's syndrome (WS) is a valuable model of accelerated ageing and results from mutations in a recQ helicase (wrn). WS fibroblasts show a mutator phenotype, replication fork stalling, increased rates of mean telomeric loss and accelerated cellular senescence. Senescence has been proposed as a candidate mechanism for the ageing of mitotic tissue. However, some mitotic tissues (such as the immune system) seem unaffected in WS. Is this evidence against a role for cell senescence in ageing? Two experiments resolve this paradox (i) the demonstration that the abbreviated replicative lifespan of WS fibroblasts can be corrected by the ectopic expression of telomerase and (ii) the demonstration that T cells derived from WS patients have the mutator phenotype characteristic of the disease but show no reduction in replicative potential. Since T cells can upregulate telomerase naturally these findings are consistent with a model in which the only wrn-mediated deletions that have a significant effect on replicative lifespan are those at or near the telomere. These data are thus supportive of a role for senescence in the ageing of the immune system. Emerging data on divisional counting mechanisms have the potential to produce many other apparent WS "paradoxes". Accordingly, we propose a general model for the phenotypic presentation of WS, which includes a modification of the Olovnikov model of telomere erosion. Somewhat unexpectedly, this predicts that accelerated senescence should not be observed in all telomerase-negative WS cell types.     

Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors

Tumor cells are usually weakly immunogenic as they largely express self-antigens and can down-regulate major histocompatability complex/peptide molecules and critical costimulatory ligands. The challenge for immunotherapies has been to provide vigorous immune effector cells that circumvent these tumor escape mechanisms and eradicate established tumors. One promising approach is to engineer T cells with single-chain antibody receptors, and since T cells require 2 distinct signals for optimal activation, we have compared the therapeutic efficacy of erbB2-reactive chimeric receptors that contain either T-cell receptor zeta (TCR-zeta) or CD28/TCR-zeta signaling domains. We have demonstrated that primary mouse CD8(+) T lymphocytes expressing the single-chain Fv (scFv)-CD28-zeta receptor have a greater capacity to secrete Tc1 cytokines, induce T-cell proliferation, and inhibit established tumor growth and metastases in vivo. The suppression of established tumor burden by cytotoxic T cells expressing the CD28/TCR-zeta chimera was critically dependent upon their interferon gamma (IFN-gamma) secretion. Our study has illustrated the practical advantage of engineering a T-cell signaling complex that codelivers CD28 activation, dependent only upon the tumor's expression of the appropriate tumor associated antigen.     

Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts

Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs. Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs. During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly 40-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 is the major CDK inhibitor for both CDK4 and CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors. Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16. p16 up-regulation is a key event in the terminal stages of growth arrest in senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.     

Glucocorticoids facilitate the stable transformation of embryonal rat fibroblasts by a polyomavirus large tumor antigen-deficient mutant.

The addition of glucocorticoids to the growth medium could substitute for the expression of the polyomavirus large tumor antigen in the transformation of rat fibroblasts in vitro. After transfection with a large tumor antigen-deficient mutant of polyomavirus, pbc1051, high-frequency permanent transformation was observed, if the cells were grown in medium containing dexamethasone. Growth of pbc1051-transfected rat fibroblasts was strictly dependent on the presence of glucocorticoids during the initial phase of transformation. In the second phase, the growth of pbc1051-transfected cells was stimulated by dexamethasone, but the hormone was not essential for growth. After approximately 10 weeks in culture, pbc1051-transfected cells had progressed to hormone independent growth. Rat embryo cells transfected with wild-type polyomavirus DNA had the second phase in which growth was stimulated by glucocorticoid, and after this phase growth was steroid independent. Addition of glucocorticoids to rat fibroblasts transfected with a plasmid encoding only the middle-sized tumor antigen resulted in only a weak stimulation of growth. In contrast, embryo cells transfected with a plasmid containing the human homologue of the cellular T24 Ha-ras gene linked to murine sarcoma virus and simian virus 40 enhancers could be efficiently established as cell lines in medium supplemented with glucocorticoids. The data suggest that, in the transformation of primary rodent cells by polyomavirus, the activity of large tumor antigen can be substituted for by stimulating normal cellular functions with dexamethasone.     

CD3 limits the efficacy of TCR gene therapy in vivo

The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/β heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.     

Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene.

Established REF52 cells (rat embryo fibroblasts) completely resist stable transformation by ras oncogenes, and simian virus 40 large tumor (T) antigen collaborates with ras to convert REF52 cells to tumorigenic state. A temperature-sensitive simian virus 40 large T antigen (encoded by tsA58) allowed the T24 Ha-ras oncogene to transform REF52 cells in a temperature-dependent manner. Two thirds of the clones transformed with tsA58 and ras became arrested in G2 or late S phase when shifted to a nonpermissive temperature for T antigen stability. Thus, ras induced growth arrest rather than stable transformation in the absence of a functional collaborating oncogene. These results indicate that collaborating oncogenes can regulate cellular responses to ras and have implications regarding therapeutic strategies to control tumor cells expressing activated ras oncogenes.     

Characterization of the absence of an unique DNA-binding protein in senescent but not in their young growing and nongrowing counterparts provides the means to mark the final stage of the cellular aging process

In senescent fibroblasts, the incapability of cell replication is permanent, and may involve the irreversible loss of gene expressions potentiating the engagement in DNA replication. The initial attempt is to identify gene products that are permanently lost in irreversibly growth-arrested cells. In this article, we report the success in identifying a DNA-associated S6 antigen found in the nuclei of growing and growth-arrested young cells, but not in the nuclei of their senescent counterparts. The presence of the S6 antigen is uniform throughout the nucleoplasm, except in the regions of the nucleoli, and found to be associated with condensed chromosomes in mitotic cells. Treatment with RNase does not abolish the antibody staining activity in the nuclei, while treatment with DNase does remove the activity. Equal intensity of S6 antibody staining is observed in transformed, growing, and contact-inhibited young fibroblasts. Significant reduced level of S6 antibody staining activity is found in the nuclei of senescent fibroblasts, indicating the loss of the expression of this protein during cellular aging process. Immunoblotting assay shows that the S6 antigen is of 50 kDa and with a possibility of a 100 kDa as a dimeric precursor. Our results suggests that a permanent turning off of unique gene expression is associated with the onset of senescence or terminal differentiation, and this hypothesis is supported by the characterization of S6 antigen's absence in in vitro-aged fibroblasts.     

Colony formation and colony size do not reflect the onset of replicative senescence in human fibroblasts

Replicative senescence of human fibroblasts in vitro has been used as a model for in vivo aging. The onset of replicative senescence varies between several months to years. A colony formation assay, critically dependent on growth speed, can be performed within weeks, and has been reported being an indicator for the onset of replicative senescence. Earlier we could not find a correlation between growth speed in mass cultures and onset of replicative senescence of human fibroblast strains. Therefore, we studied the colony formation assay in 23 fibroblast strains that varied widely in their replicative capacity. Neither the number nor the size of colonies was related to the onset of replicative senescence. The number of cells within the colonies was modestly correlated to the growth speed of the mass cultures. We conclude that the colony formation assay does not reflect the onset of replicative senescence in human fibroblasts.     

Constitutive expression of GATA-1 interferes with the cell-cycle regulation

GATA-1, mainly expressed during erythroid differentiation, has been shown to regulate the genes specifically expressed in the late stages of erythropoiesis and to protect erythroid cells from apoptosis, suggesting that it might interfere with the cell cycle. By expressing the retrovirally transduced human GATA-1 cDNA in NIH3T3 fibroblasts, we have shown that GATA-1 alone was unable to transactivate its erythroid- specific target genes in these nonerythroid cells. However, GATA-1 expression had a dramatic effect on the proliferation of these fibroblasts. The cloning efficiency of the GATA-1-expressing fibroblasts was maintained but their S phase was greatly elongated and their G1 and G2/M phases were reduced, impairing substantially their proliferation. When cultured at low serum concentrations for 48 hours, GATA-1-expressing fibroblasts failed to accumulate in the G0/G1 phases but did not become serum independent. GATA-1-expressing fibroblasts expressed D1, A, and B1 cyclin mRNAs under conditions of serum starvation or at confluence, whereas these cyclin mRNAs were downregulated in the parental NIH3T3 cells cultured under the same conditions. Moreover, these effects of GATA-1 expression on proliferation were not limited to NIH3T3 cells, since different clones of hGATA-1 virus-infected FDCP-1 cells, a murine interleukin-3- dependent hematopoietic cell line, had a slower growth rate than control cells. Based on these data, we hypothesize that GATA-1 plays a role in the regulation of the cell cycle during terminal erythroid differentiation.     

Anchorage-independent growth of normal human fibroblasts.

Normal human fibroblasts, considered to be entirely anchorage dependent for proliferation, have been grown in methylcellulose medium. The most important factor required for growth in suspension appears to be the use of high levels of serum and hydrocortisone. Newborn foreskin or fetal lung fibroblasts form colonies as large as 0.5 mm in diameter after 3 wk, with a colony-forming efficiency as high as 70%. Mouse 3T3 cells that do not form colonies in standard assays for anchorage-independent growth also grow under these conditions. Colony formation results after inoculation of as few as 100 cells per 60-mm dish, and metaphase cells have been visualized with a fluorescent DNA stain, showing that colony formation is due to division rather than aggregation. Fibroblasts recovered from suspension and grown as monolayers retain a diploid karyotype and normal shape, do not form tumors upon injection into nude mice, and become senescent. Thus, the trait of anchorage-independent growth in vitro is clearly possessed by normal human fibroblasts and can be expressed under the proper conditions.     

Suppression of tumor growth by senescence in virally transformed human fibroblasts.

Normal human cells whether embryonic, neonatal, or adult are resistant to experimentally induced tumorigenesis in contrast to rodent or chicken cells. We showed previously that neither transformation with simian virus 40 DNA nor transfection with human mutant HRAS DNA immortalized FS-2 cells (diploid, neonatal human fibroblasts). Further, tumorigenicity was not induced, despite expression of the respective transforming gene products tumor (T) antigen or p21. Here we describe treatment of FS-2 and FSSV cells with baboon endogenous virus pseudotyped Kirsten murine sarcoma virus. FSSV cells were derived from individual foci of simian virus 40-transformed FS-2 cells. The retrovirus-treated FS-2 cells (called FSK) appeared heavily granulated and expressed viral p21 but senesced during passage in culture and were not tumorigenic. The retrovirus-treated FSSV-27 cells (called FSVK-27) expressed simian virus 40 tumor antigen, had elevated levels of viral p21 protein, and formed transient tumors in nude mice. Whether grown in culture or explanted from small tumors, the FSVK-27 cells senesced. The FSVK-46 cells senesced before tumor growth occurred. On the contrary, Kirsten murine sarcoma virus (baboon endogenous virus) treatment of immortalized nontumorigenic human fibroblasts expressing simian virus 40 tumor antigen (Va2 cells) led to consistent tumor formation. The results illustrate the importance of senescence in restricting the tumor-forming ability of human cells.     

Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation.

We have constructed infectious retroviral vectors, derived from Moloney murine leukemia virus, that efficiently transduce the polyoma virus tumor (T) antigens individually. The parental vector we have chosen [pZIP-NeoSV(X)1] expresses a dominant selectable marker for neomycin resistance and is a shuttle vector capable of propagation in both eukaryotic and prokaryotic cells, thus facilitating its use in structure-function studies. To address the relationship between polyoma T-antigen tumorigenesis and the effects of individual T antigens on growth control and differentiation, we used these vectors to introduce and stably express large, middle-sized, or small T antigens into mouse fibroblasts and preadipocytes. All cDNAs introduced into the vector are expressed stably even in the absence of selective pressure. The stable expression of small T antigen is noted particularly because cell lines expressing small T antigen have not been readily available prior to the use of retroviral vectors. Small T antigen-induced increase in saturation density of NIH 3T3 cells can be scored on the basis of the morphology of drug-resistant colonies. Middle-sized T antigen eliminates the growth requirement of NIH 3T3 cells for epidermal growth factor in a defined medium and permits growth in platelet-poor plasma, indicating elimination of the platelet-derived growth factor requirement as well. Large T antigen suppresses mouse preadipocyte (3T3-F442A) differentiation. These vectors and these functional assays of T-antigen activity permit genetic analysis of the relationship between tumorigenesis by T antigens and the alteration of cellular growth and differentiation.     

Immunophenotypical changes of T lymphocytes in the elderly

BACKGROUND: Substantial changes in both representation and function of T lymphocyte subsets have been reported with advancing age. However, till now, no systematic studies focused on age-dependent changes in the expression intensity of the major T lymphocyte surface receptors. OBJECTIVE: The present study was undertaken in order to establish age-related differences in lymphocyte subpopulations by simultaneously measuring three surface antigens in young and elderly people. METHOD: Peripheral blood T cell subsets from 20 healthy elderly individuals and 15 healthy young adult donors were examined by means of a quantitative three-color flow cytometry method. RESULTS: Activated (HLA-DR+) and memory (CD45RO+) T cells, CD3+CD7- T lymphocytes, and cells expressing natural killer (NK) markers (CD3-CD56+ NK cells and CD3+CD56+ T lymphocytes) were expanded, whereas T lymphocytes expressing the adhesion molecule CD62L were lower in elderly compared with young donors. In addition to alterations in the percentages of T cell subsets during senescence, several changes in the intensity expression of T cell antigens were also detected. CD3 antigen expression was downregulated on total T lymphocytes as well as on the memory T cell subset, while CD56+ T cells exhibited increased CD3 levels. Moreover, CD2 expression, unchanged on NK cells, was upregulated on T lymphocytes from elderly subjects. CD3+CD7- T cells exhibited increased expression of CD8 antigen, while the intensity expression of HLA-DR on activated T cells and CD7 on both T and NK lymphocytes was decreased. T cells from elderly subjects also exhibited higher expression of CD50 and CD62L adhesion molecules as compared with young ones. CONCLUSION: These T cell antigen expression modulations during senescence, in addition to the alteration in the frequency of the various T lymphocyte subsets, could contribute to the complex remodeling of the immune function characteristic of the elderly.     

Syndecan expression regulates cell morphology and growth of mouse mammary epithelial tumor cells.

S115 mouse mammary epithelial cells lose their epithelial morphology and become tumorigenic when exposed to steroids. We have recently reported that testosterone exposure results in the suppression of syndecan expression, suggesting that this cell surface proteoglycan may influence S115 cell phenotype. We now report that a similar suppression and morphological response of S115 cells can be achieved by glucocorticoid exposure. We introduced into S115 cells an exogenous gene construct containing the full-length human syndecan cDNA under the control of a glucocorticoid-inducible retroviral promoter, in order to study the effect of syndecan expression on S115 cell behavior. Glucocorticoid-induced re-expression of syndecan in S115 cells restored an epithelial phenotype, while control transfectants and parental S115 cells exhibited an altered, nonepithelial phenotype. Moreover, the S115 cells expressing exogenous syndecan revealed a reduced ability to form colonies in soft agar. Therefore, the maintenance of epithelial morphology and normal growth of S115 cells are dependent on syndecan expression.     

TGF-beta signaling engages an ATM-CHK2-p53-independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells

Oncogene-induced senescence (OIS), the proliferative arrest engaged in response to persistent oncogene activation, serves as an important tumor-suppressive barrier. We show here that finite lifespan human mammary epithelial cells (HMEC) undergo a p16/RB- and p53-independent OIS in response to oncogenic RAS that requires TGF-β signaling. Suppression of TGF-β signaling by expression of a dominant-negative TGF-β type II receptor, use of a TGF-β type I receptor inhibitor, or ectopic expression of MYC permitted continued proliferation upon RAS expression. Surprisingly, unlike fibroblasts, shRNA-mediated knockdown of ATM or CHK2 was unable to prevent RAS-mediated OIS, arguing that the DNA damage response is not required for OIS in HMEC. Abrogation of TGF-β signaling not only allowed HMEC lacking p53 to tolerate oncogenic RAS but also conferred the capacity for anchorage-independent growth. Thus, the OIS engaged after dysregulated RAS expression provides an early barrier to malignant progression and is mediated by TGF-β receptor activation in HMEC. Understanding the mechanisms that initiate and maintain OIS in epithelial cells may provide a foundation for future therapies aimed at reengaging this proliferative barrier as a cancer therapy.     

Anti-ageing and rejuvenating effects of quercetin

Homeostasis is a key feature of the cellular lifespan. Its maintenance influences the rate of ageing and it is determined by several factors, including efficient proteolysis. The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Alterations of proteasome function have been recorded in various biological phenomena including ageing and replicative senescence. Proteasome activities and function are decreased upon replicative senescence, whereas proteasome activation confers enhanced survival against oxidative stress, lifespan extension and maintenance of the young morphology longer in human primary fibroblasts. Several natural compounds possess anti-ageing/anti-oxidant properties. In this study, we have identified quercetin (QUER) and its derivative, namely quercetin caprylate (QU-CAP) as a proteasome activator with anti-oxidant properties that consequently influence cellular lifespan, survival and viability of HFL-1 primary human fibroblasts. Moreover, when these compounds are supplemented to already senescent fibroblasts, a rejuvenating effect is observed. Finally, we show that these compounds promote physiological alterations when applied to cells (i.e. whitening effect). In summary, these data demonstrate the existence of naturally occurring anti-ageing products that can be effectively used through topical application.