Zhang Lab

Smurf2 regulates the senescence response and suppresses tumorigenesis in mice

Charusheila Ramkumar et al. — Fri, 04 May 2012 07:48:53 PDT

The E3 ubiquitin ligase Smurf2 mediates ubiquitination and degradation of several protein targets involved in tumorigenesis and induces senescence in human cells. However, the functional role of Smurf2 in tumorigenesis has not been fully evaluated. In this study, we generated a mouse model of Smurf2 deficiency to characterize the function of this E3 ligase in tumorigenesis. Smurf2 deficiency attenuated p16 expression and impaired the senescence response of primary mouse embryonic fibroblasts. In support of a functional role in controlling cancer, Smurf2 deficiency increased the susceptibility of mice to spontaneous tumorigenesis, most notably B cell lymphoma. At a premalignant stage of tumorigenesis, we documented a defective senescence response in the spleens of Smurf2-deficient mice, consistent with a mechanistic link between impaired senescence regulation and increased tumorigenesis. Taken together, our findings offer the genetic evidence of an important tumor suppressor function for Smurf2.

Smurf2-mediated ubiquitination and degradation of Id1 regulates p16 expression during senescence

Yahui Kong et al. — Thu, 26 Apr 2012 05:56:32 PDT

The inhibitor of differentiation or DNA binding (Id) family of transcription regulators plays an important role in cell proliferation, differentiation, and senescence. However, regulation of Id expression during these processes is poorly understood. Id proteins are known to undergo rapid turnover mediated by the ubiquitin-proteasome pathway. Anaphase-promoting complex has been shown to ubiquitinate Id2, but E3 ubiquitin ligase(s) that ubiquitinate other Id family members are not known. Here, we report for the first time the identification of Smurf2 as the E3 ligase that ubiquitinates Id1 and Id3. Smurf2-mediated ubiquitination and consequent degradation of Id1 or Id3 plays an important role in the regulation of Id expression in senescent cells. Furthermore, we found that Id1 is the mediator through which Smurf2 regulates p16 expression, providing a mechanistic link between Smurf2 and p16 expression during senescence.

Functional RNA interference (RNAi) screen identifies system A neutral amino acid transporter 2 (SNAT2) as a mediator of arsenic-induced endoplasmic reticulum stress

Raymond S. Oh et al. — Thu, 26 Apr 2012 05:56:31 PDT

Exposure to the toxic metalloid arsenic is associated with diabetes and cancer and causes proteotoxicity and endoplasmic reticulum (ER) stress at the cellular level. Adaptive responses to ER stress are implicated in cancer and diabetes; thus, understanding mechanisms of arsenic-induced ER stress may offer insights into pathogenesis. Here, we identify genes required for arsenite-induced ER stress response in a genome-wide RNAi screen. Using an shRNA library targeting ∼20,000 human genes, together with an ER stress cell model, we performed flow cytometry-based cell sorting to isolate cells with defective response to arsenite. Our screen discovered several genes modulating arsenite-induced ER stress, including sodium-dependent neutral amino acid transporter, SNAT2. SNAT2 expression and activity are up-regulated by arsenite, in a manner dependent on activating transcription factor 4 (ATF4), an important mediator of the integrated stress response. Inhibition of SNAT2 expression or activity or deprivation of its primary substrate, glutamine, specifically suppressed ER stress induced by arsenite but not tunicamycin. Induction of SNAT2 is coincident with the activation of the nutrient-sensing mammalian target of rapamycin (mTOR) pathway, which is at least partially required for arsenite-induced ER stress. Importantly, inhibition of the SNAT2 or the System L transporter, LAT1, suppressed mTOR activation by arsenite, supporting a role for these transporters in modulating amino acid signaling. These findings reveal SNAT2 as an important and specific mediator of arsenic-induced ER stress, and suggest a role for aberrant mTOR activation in arsenic-related human diseases. Furthermore, this study demonstrates the utility of RNAi screens in elucidating cellular mechanisms of environmental toxins.

Regulation of senescence in cancer and aging

Yahui Kong et al. — Wed, 23 Mar 2011 07:33:47 PDT

Senescence is regarded as a physiological response of cells to stress, including telomere dysfunction, aberrant oncogenic activation, DNA damage, and oxidative stress. This stress response has an antagonistically pleiotropic effect to organisms: beneficial as a tumor suppressor, but detrimental by contributing to aging. The emergence of senescence as an effective tumor suppression mechanism is highlighted by recent demonstration that senescence prevents proliferation of cells at risk of neoplastic transformation. Consequently, induction of senescence is recognized as a potential treatment of cancer. Substantial evidence also suggests that senescence plays an important role in aging, particularly in aging of stem cells. In this paper, we will discuss the molecular regulation of senescence its role in cancer and aging. The potential utility of senescence in cancer therapeutics will also be discussed.

O6-methylguanine induces intrachromosomal homologous recombination in human cells

Hong Zhang et al. — Wed, 23 Mar 2011 06:49:19 PDT

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which alkylates many positions in DNA including the 06 position of guanine, efficiently induces intrachromosomal homologous recombination in mouse L-cells. To investigate the role of 06-methylguanine in the induction of homologous recombination in human cells, three cell strains containing duplicated copies of the Herpes simplex virus I thymidine kinase (Htk) gene and three cell strains containing duplicated copies of the gene coding for hygromycin phosphotransferase (hyg) were treated with MNNG. Neither the Htk genes nor the hyg genes code for a functional enzyme because each contains an insertion mutation at a unique site, i.e. 8-bp XhoI linker insertions in the Htk genes and 10-bp HindIII linker insertions in the hyg genes. These cell strains differ in their level of 06-alkylguanine-DNA alkyltransferase (AGT), which specifically removes the methyl group from the 06 position of guanine. Generation of a functional Htk or hyg gene has been shown to require intrachromosomal homologous recombination between the two mutant Htk genes or the two mutant hyg genes. In all six cell strains, MNNG induced a dose-dependent increase in the frequency of homologous recombination. In each set, there was an inverse correlation between the frequency of MNNG-induced recombination and the level of AGT activity. To further study the role of 06-methylguanine in the induction of homologous recombination, we used 06-benzylguanine to inactivate AGT in two additional human cell strains containing the hyg recombination substrate. After depletion of AGT activity by 06-benzylguanine, both cell strains showed a significantly elevated level of MNNG-induced homologous recombination. These results indicate that 06-methylguanine is the principal lesion responsible for the induction of homologous recombination in these human cells by this methylating agent.

Mismatch repair is required for O(6)-methylguanine-induced homologous recombination in human fibroblasts

Hong Zhang et al. — Wed, 23 Mar 2011 06:49:17 PDT

O:(6)-methylguanine is responsible for homologous recombination induced by N:-methyl-N:'-nitro-N:-nitrosoguanidine (MNNG) [H. Zhang et al. (1996) CARCINOGENESIS:, 17, 2229]. To test the hypothesis that mismatch repair is causally involved in this process, we generated mismatch repair-deficient strains from a human fibroblast line containing a substrate for detecting intrachromosomal homologous recombination. The four strains selected for study exhibited greatly increased resistance to the cytotoxic effects of MNNG, which was not affected by depletion of O:(6)-alkylguanine-DNA alkyltransferase, and greatly increased sensitivity to the mutagenic effect of MNNG, suggesting that the mutagenic base modifications induced in these four cell strains by MNNG persist in their genomic DNA. Tests showed that their extracts are deficient in the repair of G:T mismatches. The frequency of homologous recombination induced by MNNG in three of these strains was significantly (5-7-fold) lower than that induced in the parental cell strain. This was not the result of a generalized defect in recombination, because when (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epox y-7,8,9, 10-tetrahydrobenzo[a]pyrene was used to induce recombination, all three lines responded with a normal or even a somewhat higher frequency than that observed in the parental strain. The lack of recombination displayed by the fourth strain was shown to result from the loss of part of the recombination substrate. The results strongly suggest that functional mismatch repair is required for MNNG-induced homologous recombination.

hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

Ziqiang Li et al. — Wed, 23 Mar 2011 06:49:16 PDT

In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not possible to demonstrate that the decrease in mutagenesis reflected decreased hRev3 protein. However, the conclusion is supported by the fact that in a similar study with a strain expressing a high level of antisense hREV1, a very similar result was obtained, i.e. UV or BPDE mutagenesis was virtually eliminated.

Senescence-specific gene expression fingerprints reveal cell-type-dependent physical clustering of up-regulated chromosomal loci

Hong Zhang et al. — Wed, 23 Mar 2011 06:49:15 PDT

Replicative senescence is the state of irreversible proliferative arrest that occurs as a concomitant of progressive telomere shortening. By using cDNA microarrays and the gabriel system of computer programs to apply domain-specific and procedural knowledge for data analysis, we investigated global changes in gene transcription occurring during replicative senescence in human fibroblasts and mammary epithelial cells (HMECs). Here we report the identification of transcriptional "fingerprints" unique to senescence, the finding that gene expression perturbations during senescence differ greatly in fibroblasts and HMECs, and the discovery that despite the disparate nature of the chromosomal loci affected by senescence in fibroblasts and HMECs, the up-regulated loci in both types of cells show physical clustering. This clustering, which contrasts with the random distribution of genes down-regulated during senescence or up-regulated during reversible proliferative arrest (i.e., quiescence), supports the view that replicative senescence is associated with alteration of chromatin structure.

Disparate effects of telomere attrition on gene expression during replicative senescence of human mammary epithelial cells cultured under different conditions

Hong Zhang et al. — Wed, 23 Mar 2011 06:49:13 PDT

Telomere shortening in populations of human mammary epithelial cells (HMECs) that survive early replicative arrest (M0) by the inactivation of p16(INK4A) during cell culture on plastic dishes leads to a state of permanent replicative arrest termed senescence. While culture of HMECs on feeder layers abrogates M0 and p16(INK4A) inactivation, progressive telomere attrition in these cells also eventually results in permanent replicative arrest. Expression of telomerase prevents both senescence on plastic (S-P) and senescence on feeder layers (S-FL) in HMECs, as it does also in cultured primary human fibroblasts. We report here that the gene expression profiles of senescence in HMECs of the same lineage maintained under different culture conditions showed surprisingly little commonality. Moreover, neither of these senescence-associated profiles in HMECs resembles the profile for senescence in human fibroblasts. These results indicate that senescence-associated alterations in gene expression resulting from telomere attrition are affected by culture conditions as well as by cell origins, and argue that replicative senescence at the molecular level is a diverse rather than unique cellular process.

Smurf2 up-regulation activates telomere-dependent senescence

Hong Zhang et al. — Wed, 23 Mar 2011 06:49:11 PDT

Progressive telomere shortening activates replicative senescence, which prevents somatic cells from being propagated indefinitely in culture. The limitation of proliferative capacity imposed by replicative senescence is thought to contribute to both organismal aging and the prevention of tumor development. Here we report that up-regulation of Smurf2, an E3 ubiquitin ligase previously implicated in TGF-beta signaling, is a specific consequence of telomere attrition in human fibroblasts and that such up-regulation is sufficient to produce the senescence phenotype. Adventitious production of the Smurf2 protein in early passage fibroblasts at the same physiological level observed during telomere-mediated senescence resulted in proliferative arrest in a viable state, morphological and biochemical alterations characteristic of senescence, acquisition of senescence-specific alterations in gene expression, and reversal of cellular immortalization by telomerase. We show that the senescence-inducing actions of Smurf2 occur in the absence of detectable DNA damage or stress response, that Smurf2's effects require a novel function distinct from its E3 activity, that Smurf2 recruits the Rb and p53 pathways for senescence induction, and that while p21 is elevated by Smurf2, Smurf2-mediated senescence is independent of p21. Smurf2 is the first gene found to be both up-regulated by telomere attrition and sufficient to induce senescence.

Molecular signaling and genetic pathways of senescence: Its role in tumorigenesis and aging

Hong Zhang — Wed, 23 Mar 2011 06:49:10 PDT

In response to progressive telomere shortening in successive cell divisions, normal somatic cells enter senescence, during which they cease to proliferate irreversibly and undergo dramatic changes in gene expression. Senescence can also be activated by various types of stressful stimuli, including aberrant oncogenic signaling, oxidative stress, and DNA damage. Because of the limited proliferative capacity imposed by senescence, as well as the ability of senescent cells to influence neighboring non-senescent cells, senescence has been proposed to play an important role in tumorigenesis and to contribute to aging. Considerable effort has been put into elucidating the molecular mechanisms of senescence, including the signals that trigger senescence, the molecular pathways by which cells enter senescence, and evidence that supports its role in tumorigenesis and aging.

Suppression of human tumor cell proliferation by Smurf2-induced senescence

Hong Zhang et al. — Wed, 23 Mar 2011 06:49:08 PDT

The limitation of proliferative potential in human somatic cells imposed by replicative senescence has been proposed as a mechanism of tumor suppression. The E3 ubiquitin ligase Smurf2 is up-regulated during replicative senescence in response to telomere shortening, and induces senescence when expressed adventitiously in early passage or telomerase-immortalized human fibroblasts. To investigate the generality of Smurf2's control of cell proliferation, we have studied the effects of Smurf2 up-regulation on cell proliferation in early passage human mammary epithelial cells which normally do not show elevated expression of Smurf2 during senescence, and in 16 human cancer cell lines derived from both sarcomas and carcinomas. Here we report that Smurf2 up-regulation induced senescence in a wide variety of human cell types, including highly neoplastic cell lines. Consistent with our previous findings, the ability of Smurf2 to arrest cell proliferation did not require its ubiquitin ligase activity. Furthermore, expression of the cyclin-dependent kinase inhibitor p21 was increased in tumor cells undergoing Smurf2-induced senescence, and such increase occurred independently of the transactivation function of p53. Our results, which reveal a previously unsuspected tumor suppression function for Smurf2-induced senescence, suggest that modulation of Smurf2 action may be a useful strategy for inhibition of cancer cell growth.