Androgen receptor levels are increased by interferons in human prostate stromal and epithelial cells.

Proliferation of normal and malignant prostate epithelium is regulated by androgen stimulation via both the androgen receptor (AR)-positive stromal and epithelial cells. However, it is not known how AR expression is regulated in human prostate cells. We report that treatment of normal human prostate stromal cells (PrSCs) with type I IFN (alpha or beta), but not type II IFN (gamma), resulted in increased levels of AR protein. The maximal increase in AR protein levels was dependent on the dose and the duration of the IFN-alpha treatment. We found that the increase in AR protein levels was independent of de novo transcription and protein synthesis. Interestingly, the IFN-alpha treatment of PrSCs resulted in considerable nuclear accumulation of AR, stimulation of AR-mediated transcription of reporter genes, and retardation of cell proliferation. Furthermore, treatment of normal human prostate epithelial cells with IFNs (alpha, beta or gamma) also resulted in increased levels of AR protein. Together, our observations identify the androgen receptor as an IFN-regulated protein in normal human prostate stromal and epithelial cells and predict that IFN-induced levels of AR in prostate cells contribute to the regulation of androgen signaling.

Resistance to UV-induced apoptosis in human keratinocytes during accelerated senescence is associated with functional inactivation of p53.

Compared to proliferating keratinocytes (KCs), growth-arrested KCs are relatively resistant to UV-light induced apoptosis. When KCs undergo confluency, or following exposure to anti-proliferative agents such as IFN-gamma plus a phorbol ester-12-O-tetradecanoylyphorbol-13-acetate (TPA), they convert from a proliferative to a nonproliferative state resembling senescence. Since p53 regulates UV-induced apoptosis of KCs, this report further characterizes p53 half-life, post-translational modifications, and transcriptional activity using cultured human KCs and living epidermal equivalents. The half-life of p53 in KCs was longer than fibroblasts (greater than approximately 3 h vs. 30 min). Exposure of proliferating KCs to UV-light induces post-translational modifications of p53 including acetylation of lysine-382 residues. By contrast, KCs undergoing irreversible growth arrest following confluency, or exposure to IFN-gamma plus TPA, were resistant to UV-induced apoptosis, and failed to undergo the acetylation modification of p53. Exposure of KCs to IFN-gamma plus TPA reduced total cellular p53 levels and reduced the transcriptional activity of p53. Addition of Trichostatin A (TSA), an inhibitor of de-acetylation, increased acetylation of lysine-382 in confluent KCs, thereby enhancing susceptibility of confluent cultures to UV-induced apoptosis. Pre-treatment of epidermal equivalents with IFN-gamma plus TPA also blocked UV-light induced increase in p53 levels, and reduced apoptosis. In conclusion, these studies demonstrate that growth arrested KCs may resist UV-light induced apoptosis by inactivating the pro-apoptotic function of p53.

p202, an interferon-inducible negative regulator of cell growth, is a target of the adenovirus E1A protein.

Studies have revealed that human adenovirus-encoded E1A protein promotes cell proliferation through the targeted interaction with cellular proteins that act as key negative regulators of cell growth. The targets of E1A protein include the retinoblastoma tumor suppressor protein (pRb). Because p202, an interferon (IFN)-inducible murine protein (52-kDa), negatively regulates cell growth in part through the pRb/E2F pathway, we tested whether the p202 is a target of the adenovirus-encoded E1A protein for functional inactivation. Here we report that the expression of E1A protein overcame p202-mediated inhibition of cell growth and this correlated with an alleviation of p202-mediated inhibition of the transcriptional activity of E2F. Furthermore, E1A protein relieved p202-mediated inhibition of the specific DNA-binding activity of E2F complexes, including those containing the pocket proteins. Additionally, the E1A protein bound to p202 both in vitro and in vivo and a deletion of four amino acids in the conserved region 2 (CR2) of E1A protein significantly reduced the binding of E1A to p202. Interestingly, ectopic expression of p202 under reduced serum conditions significantly reduced E1A-mediated apoptosis. Taken together, our observations provide support to the idea that the p202 and adenovirus E1A protein functionally counteract each other and E1A protein targets p202 to promote cell proliferation.

Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus.

The Nba2 locus is a major genetic contribution to disease susceptibility in the (NZB x NZW)F(1) mouse model of systemic lupus. We generated C57BL/6 mice congenic for this NZB locus, and these mice produced antinuclear autoantibodies characteristic of lupus. F(1) offspring of congenic and NZW mice developed high autoantibody levels and severe lupus nephritis similar to (NZB x NZW)F(1) mice. Expression profiling with oligonucleotide microarrays revealed only two differentially expressed genes, interferon-inducible genes Ifi202 and Ifi203, in congenic versus control mice, and both were within the Nba2 interval. Quantitative PCR localized increased Ifi202 expression to splenic B cells and non-T/non-B cells. These results, together with analyses of promoter region polymorphisms, strain distribution of expression, and effects on cell proliferation and apoptosis, implicate Ifi202 as a candidate gene for lupus.

Abnormal NF-kappaB signaling pathway with enhanced susceptibility to apoptosis in immortalized keratinocytes.

The transcriptional activation and proper regulation of NF-kappaB is known to be important to the apoptotic resistant phenotype of epidermal-derived keratinocytes. By comparing and contrasting the responses of normal foreskin-derived keratinocytes versus an immortalized skin-derived keratinocyte cell line (i.e. HaCaT cells), several molecular defects involving NF-kappaB signaling pathway were delineated in the immortalized keratinocytes. While exposure to IFN-gamma plus TPA produces growth arrest in both normal and immortalized keratinocytes, with rapid phosphorylation of MEKKI and recruitment of distinctive protein kinase C isoforms into the signalosome complex, subsequent molecular events necessary for NF-kappaB activation were abnormal in HaCaT cells. This disrupted NF-kappaB activation in HaCaT cells was accompanied by enhanced susceptibility to UV-light induced apoptosis, which was associated with elevated levels of E2F-1 and decreased TRAF1/TRAF2 levels. Additional defects in HaCaT cells included markedly diminished levels of IKKbeta (and lack of induction of kinase activity) in response to inflammatory stimuli, a failure of p21(WAF1/CIP1) to associate with CDK2, and a decreased association between p65 and p300. These studies suggest caution in using HaCaT cells as a substitute for normal keratinocytes to study apoptosis in the skin. Thus, it appears that while the immortalized cells can escape cell cycle checkpoints by elevated levels of E2F-1, an adverse biological consequence of such dysregulated cell cycle control is the inability to activate the anti-apoptotic NF-kappaB signaling pathway. Therefore, exploiting this apoptosis vulnerability in pre-malignant, or immortalized cells, prior to acquiring a death-defying phenotype characteristic of more advanced malignant cell types, provides the basis for an early interventional therapeutic strategy for cutaneous oncologists.

The gene encoding p202, an interferon-inducible negative regulator of the p53 tumor suppressor, is a target of p53-mediated transcriptional repression.

The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We reported previously that overexpression of p202, an interferon-inducible negative regulator of cell growth, negatively regulates the transcriptional activity of p53. Now we identify the gene encoding p202 as one whose mRNA and protein expression decrease in cells following the expression of wild-type, but not mutant, p53. Furthermore, the levels of p202 also decrease after exposure of cells to ultra violet light, which correlate with increase in the levels of p53. We report that the sequence-specific DNA binding of p53 to the 5'-regulatory region of the 202 gene contributes to the transcriptional repression of the 202 gene. Interestingly, overexpression of p202 in cells induced to undergo p53-dependent apoptosis significantly delays this process, indicating that the negative regulation of the 202 gene by wild-type p53 is important to potentiate apoptosis.

p202 levels are negatively regulated by serum growth factors.

p202 is an IFN-inducible phosphoprotein (Mr 52,000) whose expression in transfected cells retards proliferation. Interestingly, the reduced levels of p202 in fibroblasts (in consequence of the expression of antisense to 202 RNA), under reduced serum conditions, increase the susceptibility of cells to apoptosis. To identify the functional role of p202 in cell growth regulation, we tested whether serum growth factor levels in the culture medium affect p202 levels. Here we report that, under reduced serum conditions, the p202 levels were increased in fibroblasts, and the increase was seen at both the mRNA and protein levels. Moreover, an increase in p202 levels was correlated with cell growth arrest in the G1 phase of the cell cycle. Interestingly, the presence of platelet-derived growth factor AB, basic fibroblast growth factor, or transforming growth factor beta1 in the culture medium abrogated the increase in p202 levels seen under reduced serum conditions. We found that the increase in p202 levels was accompanied by an increase in JunD/activation protein 1(AP-1) levels, and transfection of a JunD-encoding plasmid along with a reporter plasmid in which transcription of the reporter gene (luciferase) was driven by the 5'-regulatory region of the 202 gene resulted in an increase in the activity of luciferase. Additionally, stable overexpression of JunD in cells, under reduced serum conditions, also resulted in an increase in p202 levels. Interestingly, one of the AP-1-like DNA-binding sequences present in the 5'-regulatory region of the 202 gene could selectively bind to the JunD/AP-1 transcription factor. Taken together, our observations reported herein suggest that in fibroblasts, under reduced serum conditions, the increased levels of JunD/AP-1 contribute to the transcriptional up-regulation of p202 levels, which may be important for the regulation of apoptosis.

Cytoplasmic localization of the interferon-inducible protein that is encoded by the AIM2 (absent in melanoma) gene from the 200-gene family.

While interferons (IFNs) (alpha, beta and gamma), a family of cytokines, have the ability to exert the growth-inhibitory effect on target cells, the molecular mechanism(s) by which IFNs inhibit cell growth remains to be identified. Because IFN-inducible 'effector' proteins mediate the biological activities of IFNs, characterization of IFN-inducible proteins is critical to identify their functional role in IFN action. One family (the 200-family) of IFN-inducible proteins is encoded by structurally related murine (Ifi202a, Ifi202b, Ifi203, Ifi204 and D3) and human (IFI16, MNDA and AIM2) genes. The proteins encoded by genes in the family share a unique repeat of 200-amino acids and are primarily nuclear. The AIM2 gene is a newly identified gene that is not expressed in a human melanoma cell line. Here we report that AIM2 is estimated to be a 39 kDa protein and, unlike other proteins in the family, is localized primarily in the cytoplasm. Interestingly, overexpression of AIM2 in transfected cells retards proliferation and, under reduced serum conditions, increases the susceptibility to cell death. Moreover, AIM2 can heterodimerize with p202 in vitro. Together, these observations provide support to the idea that AIM2 may be an important mediator of IFN action.

Differential induction of the 200-family proteins in Daudi Burkitt's lymphoma cells by interferon-alpha.

Interferon (IFN)-inducible "effector" proteins mediate the biological activities of the IFNs. Therefore, the identification of the functional role(s) of IFN inducible proteins in IFN action is important to elucidate the molecular mechanisms by which IFNs inhibit cell growth. One family (the "200-family") of IFN-inducible proteins includes structurally related murine (p202a, p202b, p203, p204 and D3) and human (MNDA, IFI-16 and AIM2) proteins. However, their role in IFN action remains to be established. Here we report that IFN-alpha treatment of Daudi Burkitt's lymphoma cells resulted in differential induction of MNDA, IFI 16, and a p202-related protein (p202RP). Interestingly, IFN induction of p202RP preceded the induction of MNDA and IFI 16 proteins and the growth inhibition by IFN. Additionally, the induction of these proteins by IFN was accompanied by: (i) a transient increase in p21(WAF1/CIP1) levels; (ii) an increase in the functional form of pRb and p130; (iii) an inhibition of the sequence-specific DNA binding activity of E2F complexes; and (iv) a marked decrease in c-Myc levels. Our observations reported herein provide support to the hypothesis that IFN-inducible p202RP and MNDA proteins from the 200-family contribute to the growth inhibitory activities of the IFNs.

Role of NF-kappaB in the apoptotic-resistant phenotype of keratinocytes.

Several studies point to a role for NF-kappaB in modulating epidermal thickness and apoptotic susceptibility of keratinocytes. When phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) are topically applied, prominent epidermal thickening occurs, and exposure to interferon (IFN)-gamma promotes increased epidermal thickness producing psoriatic lesions. While keratinocytes derived from psoriatic plaque resist apoptosis, and combination of TPA and IFN-gamma activates NF-kappaB, the molecular mechanism linking NF-kappaB activation and keratinocyte apoptosis resistance was unknown. Therefore, we examined the ability of IFN-gamma plus TPA to influence NF-kappaB activity, gene expression, and response to UV light-induced apoptosis. These responses in normal keratinocytes were compared with immortalized keratinocytes (HaCaT cells). Exposure of normal keratinocytes to IFN-gamma plus TPA produced a synergistic activation of NF-kappaB, compared with when each reagent was used individually. Normal keratinocytes when exposed to IFN-gamma plus TPA acquired a resistance to UV light-induced apoptosis, which was dependent on NF-kappaB because expression of a dominant negative form of IkappaBalpha overcame the resistance. Compared with normal keratinocytes, HaCaT cells have a dysfunctional constitutive NF-kappaB signaling pathway not induced by IFN-gamma and TPA, rendering HaCaT cells highly susceptible to UV-induced apoptosis. Thus, immortalized HaCaT cells have an abnormal constitutive and dysfunctional NF-kappaB signaling system. These results provide evidence that activation and proper regulation of NF-kappaB is essential for acquisition of an apoptotic-resistant phenotype for epidermal-derived keratinocytes.

Apoptosis in proliferating, senescent, and immortalized keratinocytes.

Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-kappaB were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-kappaB is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-kappaB, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-kappaB but not early differentiation.

Retardation of cell proliferation after expression of p202 accompanies an increase in p21(WAF1/CIP1).

p202 is an IFN-inducible, primarily nuclear, phosphoprotein (52-kDa) whose constitutive overexpression in transfected cells inhibits colony formation. To investigate the molecular mechanism(s) by which expression of p202 protein impairs colony formation, we established stable cell lines that inducibly express p202. Using this cell model, we demonstrate that the induced expression of p202 in asynchronous cultures of these cells was accompanied by: (a) an increase in steady-state levels of p21(WAF1/CIP1/SDI1) (p21) mRNA and protein; (b) a decrease in Cdk2 protein kinase activity; and (c) an increase in the functional form of retinoblastoma protein (pRb). Transient transfection of a p202-encoding plasmid in Saos-2 cells, which do not harbor a wild-type p53 protein, resulted in an increase in p21 protein, which indicated that p202 could regulate expression of p21 protein independent of p53 protein. Moreover, we demonstrate that expression of p202 in these cells increased cell doubling time without accumulation of cells in a particular phase of the cell cycle. Taken together, these results are consistent with the possibility that p202 protein contributes to the cell growth retardation activity of the IFNs, at least in part, by modulating p21 protein levels.

Reduced growth rate and transformation phenotype of the prostate cancer cells by an interferon-inducible protein, p202.

Interferons (IFNs) can exert cytostatic and immunomodulatory effects on carcinoma cells. In particular, growth inhibition of human prostate carcinoma by IFNs has been demonstrated both in vitro and in vivo. p202 is a 52 kd nuclear phosphoprotein known to be induced by IFNs. In this report, we showed that the expression of p202 was associated with an anti-proliferative effect on human prostate cancer cells. More importantly, cells that expressed p202 showed reduced ability to grow in soft-agar, indicating a loss of transformation phenotype. Our data suggest that p202 is a growth inhibitor gene in prostate cancer cells and its expression may also suppress transformation phenotype of prostate cancer cells.

p202 self-associates through a sequence conserved among the members of the 200-family proteins.

Murine p202 is an interferon-inducible primarily nuclear phosphoprotein (52 kDa) whose expression in transfected cells inhibits colony formation. p202-binding proteins include the pocket proteins (pRb, p107 and p130), a p53-binding protein (sm53BP1), and transcription factors (e.g. NF-kappaB (p50 and p65), AP-1 (c-Fos and c-Jun), E2F-1, E2F-4, MyoD, and myogenin). p202 modulates the transcriptional activity of these factors in transfected cells. Here we demonstrate that p202 self-associates directly and a sequence in p202, which is conserved among the members of the 200-family proteins, was sufficient for self-association in vitro. Our observations reported herein raise the possibility that self-association of p202 may provide a mechanism for the regulation of its activity.

p202 prevents apoptosis in murine AKR-2B fibroblasts.

p202 is an interferon (IFN)-inducible, primarily nuclear, phosphoprotein (52-kDa) whose overexpression in transfected cells inhibits colony formation. p202 binds to the retinoblastoma tumor suppressor protein and two other members of the pocket family proteins (p107 and p130). Moreover, overexpression of p202 in transfected cells inhibits the transcriptional activity of E2Fs (E2F-1/DP-1 and E2F-4/DP-1), p53, AP-1 c-Fos and c-Jun, NF-kappaB p50 and p65. Here we demonstrate that inhibition of endogenous p202 production in murine AKR-2B fibroblasts did not result in an increase in cell proliferation. Instead, these cells exhibited increased susceptibility to apoptosis in response to decrease in serum concentrations in the growth medium. These observations are consistent with the notion that normal levels of p202 may be needed for the regulation of cell proliferation.

Inhibition of E2F-4/DP-1-stimulated transcription by p202.

The interferon (IFN)-inducible proteins mediate activities of the interferons including the cell growth-regulatory activity. We have shown that p202, an IFN-inducible 52kDa primarily nuclear phosphoprotein whose expression in transfected cells inhibits cell proliferation, interacts with the retinoblastoma tumor suppressor protein (pRb) and the transcription factor E2F (E2F-1/ DP-1) in vitro and in vivo. p202 was shown to inhibit E2F-1/DP-1-stimulated transcription of a reporter gene and of endogenous genes. Here we report that expression of p202 inhibited E2F-4/DP-1-stimulated transcription of a reporter gene in transfected cells. Furthermore, this inhibition was associated with the inhibition of the sequence-specific DNA-binding of E2F-4 both in complex with the pocket proteins p107 or p130 and in its 'free' form in vitro. p202 bound to p107 and p130 in vitro and in vivo and also associated with E2F-4, supporting the notion that complexes containing p107/E2F-4 or p130/ E2F-4 and p202 exist in vivo. Moreover, cotransfection of E2F-4-encoding plasmid in AKR-2B cells overcame p202-mediated inhibition of cell growth, raising the possibility that p202 contributes to cell growth inhibition by the interferons, at least in part, by modulating E2F-4-mediated transcription.

p202, an interferon-inducible modulator of transcription, inhibits transcriptional activation by the p53 tumor suppressor protein, and a segment from the p53-binding protein 1 that binds to p202 overcomes this inhibition.

p202, an interferon-inducible murine protein, is a member of the "200 family" of proteins and is primarily nuclear. p202 is a modulator of transcription; it binds several transcription factors, including NF-kappaB p50 and p65, AP-1 c-Fos and c-Jun, and E2F1, and inhibits their transcriptional activity. p202 also binds pRb, the retinoblastoma protein, and if overexpressed it retards cell proliferation. Here we report that using the yeast two-hybrid assay we found that p202 bound the murine homolog of the human p53-binding protein 1 (53BP1), a protein shown to interact with the DNA binding domain of the p53 tumor suppressor protein. p202 bound a 98amino acid segment from 53BP1. This binding was inhibited by the replacement in p202 of a histidine (from the M(F/L)HATVA(T/S) sequence that is conserved among all of the 200 family proteins) by phenylalanine. We also report that overexpression of p202 inhibited the p53-dependent expression of reporter genes containing p53-activable segments from the mdm2 and p21 genes, whereas a decrease in the p202 level (in consequence of the expression of 202 antisense RNA) resulted in an increase in the p53-dependent expression of these reporters. Expression of the 53BP1 segment binding to p202 overcame the inhibition by overexpressed p202 of the transcription of reporters mediated by the p53 or the AP-1 transcription factors and of the proliferation of yeast.