Protein Levels Of PARP Research Articles

Abstract 2518: TAK-733 is a Potent and Selective Inhibitor of Mammalian MEK Kinases Capable of Inducing Apoptosis in BRAF Driven Cells Both in vitro and in vivo

Abstract Mutations in BRAF a serine/threonine protein kinase are frequently observed in human cancers. A valine to glutamic acid mutation at amino acid position 600 accounts for nearly 80% of all the activating mutations found for this kinase. This mutation (V600E) forces BRAF to become constitutively active and phosphorylate its substrates regardless of upstream signaling. Numerous cancers in tissues such as skin, colon, thyroid, and ovary have been attributed to uncontrolled proliferation driven by this mutation. TAK-733 represents a novel and distinct chemotype which is capable of allosteric inhibition of the BRAF substrates MEK-1 and MEK-2 at low nanomolar concentrations. Viability assays in established human cancer cell lines which bear the BRAF V600E mutation demonstrate this compound was also capable of lowering NADH levels with single digit nanomolar EC50s. Loss of NADH production can be attributed to the induction of programmed cell death pathways involving Caspases 3 and 7. Induction of these apoptosis markers was confirmed in vitro by a Caspase-GLO assay. TAK-733 induces apoptosis more potently in COLO 205 (BRAF V600E) human colo-rectal carcinoma cells when compared to other MEK inhibitors. Pharmacodynamic analysis of COLO 205 tumor bearing mice confirmed TAK-733, unlike other MEK inhibitors tested, is capable of inducing apoptosis 36 hours after a single oral administration. To determine whether the anti-tumor effect of TAK-733 is associated with apoptosis, A375 (BRAF V600E) human melanoma tumors were harvested at sacrifice and protein extracted to determine cleavage products of PARP. A single oral treatment with 1, 10, and 30 mg/kg TAK-733 increased protein expression of cleaved PARP in a dose and time dependent manner. Elevated cleaved PARP protein expression was detected as early as 0.5-hours in tumors following 30 mg/kg dose and detectable at all groups by 4-hours. The increase cleaved PARP protein levels persisted for at least 24-hours and returned to control expression levels by 48-hours. Daily oral administration of 1, 10, and 30 mg/kg/day TAK-733 for 14 days potently inhibited tumor growth resulting in tumor growth delay of 3.4, 10.8, and 26.6 days in A375 cell implanted mice. A 60% partial regression response rate (tumor volume reduced by more than 50% of initial volume) was observed in mice that received 30 mg/kg/day TAK-733. These data suggest a possible explanation as to why TAK-733 is capable of inducing regressions in COLO 205 and A375 tumor growth inhibition studies, and may represent a differentiation point from other MEK inhibitors currently in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2518.

Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells

BackgroundThe progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells.MethodsFollowing exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay.ResultsDCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1/2- and p38 MAPK while Erk1/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure.ConclusionDCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate.

Mitochondrial DNA Depletion Reduces PARP-1 Levels and Promotes Progression of the Neoplastic Phenotype in Prostate Carcinoma

Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) mutations and/or depletion has been correlated with cancer progression and drug resistance. To investigate the role of mtDNA in prostate cancer progression, we used LNCaP and PC-3 prostate carcinoma cells as experimental model. Compared to minimally invasive androgen-dependent LNCaP cells, highly invasive androgen-independent PC-3 cells, as well as androgen-independent DU145 and C4-2 cells, exhibited significantly reduced mtDNA content. In PC-3 cells, reduction of mtDNA was accompanied by decreased mitochondrial membrane potential (ΔΨm), increased migration onto the basement membrane protein laminin-1, reduced chemosensitivity to paclitaxel (IC50=110 nM vs. 22 nM) and decreased expression of poly(ADP-ribose) polymerase (PARP)-1. To investigate the relationship between mtDNA depletion and these phenotypic characteristics, we established mtDNA-depleted LNCaP cells [Rho(−)] by long-term exposure to ethidium bromide or treated wild-type LNCaP cells with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone. Both manipulations resulted in ΔΨm loss, acquisition of invasive cytology, increased motility onto laminin-1, reduced sensitivity to paclitaxel (IC50=~100 nM) and ~75% reduction in PARP-1 protein levels, resembling PC-3 cells. Overall, these results provide novel evidence demonstrating that mtDNA depletion in early prostate carcinoma may contribute to the acquisition of a more invasive phenotype that is less sensitive to paclitaxel-induced apoptosis.

Mitochondrial DNA depletion reduces PARP-1 levels and promotes progression of the neoplastic phenotype in prostate carcinoma.

Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) mutations and/or depletion has been correlated with cancer progression and drug resistance. To investigate the role of mtDNA in prostate cancer progression, we used LNCaP and PC-3 prostate carcinoma cells as experimental model. Compared to minimally invasive androgen-dependent LNCaP cells, highly invasive androgen-independent PC-3 cells, as well as androgen-independent DU145 and C4-2 cells, exhibited significantly reduced mtDNA content. In PC-3 cells, reduction of mtDNA was accompanied by decreased mitochondrial membrane potential (ΔΨm), increased migration onto the basement membrane protein laminin-1, reduced chemosensitivity to paclitaxel (IC50=110 nM vs. 22 nM) and decreased expression of poly(ADP-ribose) polymerase (PARP)-1. To investigate the relationship between mtDNA depletion and these phenotypic characteristics, we established mtDNA-depleted LNCaP cells [Rho(−)] by long-term exposure to ethidium bromide or treated wild-type LNCaP cells with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone. Both manipulations resulted in ΔΨm loss, acquisition of invasive cytology, increased motility onto laminin-1, reduced sensitivity to paclitaxel (IC50=~100 nM) and ~75% reduction in PARP-1 protein levels, resembling PC-3 cells. Overall, these results provide novel evidence demonstrating that mtDNA depletion in early prostate carcinoma may contribute to the acquisition of a more invasive phenotype that is less sensitive to paclitaxel-induced apoptosis.

Apoptosis, cell cycle progression and gene expression in TP53-depleted HCT116 colon cancer cells in response to short-term 5-fluorouracil treatment

Loss of TP53 function may contribute to 5-fluorouracil (5-FU) resistance in colorectal cancer since TP53-deficient cells may be unable to undergo apoptosis in response to 5-FU-induced DNA damage. 5-FU treatment of TP53-deficient cells would provide useful information on the apoptotic response to drug-induced DNA damage in the absence of TP53 and its transcriptional targets. We investigated apoptosis induction and cell cycle alterations in response to short-term treatment with two different 5-FU concentrations following siRNA-mediated knockdown of TP53 in the TP53-proficient HCT116 colon cancer cell line. We focused on high-dose 5-FU treatment to investigate the apoptotic phenotype in 5-FU-treated cultures since this dose resulted in apoptosis induction at 24 h of treatment, whereas clinically-relevant bolus 5-FU treatment of HCT116 cultures did not. Gene expression alterations were also assessed in 5-FU-treated HCT116 cultures using whole genome expression arrays. Compared to 5-FU-treated TP53-proficient HCT116 cultures, 5-FU-treated TP53-depleted HCT116 cultures showed lack of CDKN1A induction, decreased apoptotic levels, decreased FAS and TNFRSF10B transcript levels and cleaved PARP protein levels, G1/S transition arrests, decreased CCND1 protein levels, and smaller intra-S phase arrests. Alterations in gene expression in 5-FU-treated TP53-depleted HCT116 cultures confirmed previously-reported TP53 target genes and suggested potentially novel TP53 target genes (e.g. APOBEC3C, BIRC3, JMJD2B, LAMP3, MYO1E, PRRG1, SULF2, TACSTD2, TncRNA, ZFYVE20) that may play a role in mediating the 5-FU-induced DNA damage response in TP53-proficient cells. Abrogation of TP53 function in 5-FU-treated HCT116 cultures results in reduced apoptosis, TP53- and CDKN1A-independent G1/S phase arrests that may be protective against apoptosis, smaller intra-S phase arrests, and transcript level decreases of both reported TP53 target genes as well as potentially novel TP53 target genes.

Preliminary Study on Effects of Marsdenia Tenacissima Injection on NB4 Cells.

Purpose To study the effect of Marsdenia tenacissima injection on NB4 leukemia cells and its mechanisms.Methods After treated with Marsdenia tenacissima, viability of NB4 leukemia cells were determined by trypan blue assay. Cell cycle was assessed by FACS. Cell differentiation were determined by cell morphology, cell surface differentiation antigen and NBT reduction test. Cell cycle and apoptosis associated proteins were measured by western blot.Results Marsdenia tenacissima inhibits the growth of leukemia cells in a dose and time dependent manner. Marsdenia tenacissima induces G0/G1 phase arrest of NB4 cells, accompanied by P21 up-regulation. Marsdenia tenacissima did not change the protein level of PARP and p27. Marsdenia tenacissima could increase the expression of CD11b and percent of NBT positive cells, induces differentiation of NB4 cells.Conclusion Marsdenia tenacissima could inhibit the proliferation of NB4 cells through inducing cell cycle arrest and cell differentiation.

Survivin Inhibition of p34Cdc2 Via Protection of Wee1 Correlates with Survival.

The inhibitor of apoptosis protein Survivin and p34Cdc2 (the cyclin-dependent kinase, Cdk1) are involved in cell cycle progression and apoptosis. Cdc2 and Survivin are tightly regulated in normal cells but deregulated in cancer. We have previously shown that Survivin regulates entry of hematopoietic stem cells into cell cycle. It has been established that Cdc2 phosphorylates Survivin at threonine 34 (T34), which is believed to be a stabilizing event necessary for normal Survivin function and anti-apoptotic activity. Activation of Cdc2 is required for its apoptotic activity and inhibition of the proapoptotic activity of Cdc2 can be achieved by phosphorylation at tyrosine 15 (Tyr15). Stable overexpression of a wild-type (wt) mouse Survivin-IRES-GFP construct in IL-3-dependent mouse BaF3 cells resulted in increased Tyr15 phosphorylation of Cdc2 and enhanced cell proliferation, while cells transduced with a threonine 34 to alanine (T34A) dominant negative (DN) Survivin construct showed reduced Cdc2 Tyr15 phosphorylation and accelerated apoptosis. Furthermore, when apoptosis was induced in stably transduced BaF3 cells by IL-3 withdrawal, Survivin expression and phospho-Tyr15 levels correlated with enhanced survival and decreased survival and low phospho-Tyr15 levels correlated with Survivin disruption by T34A-DN-Survivin. Since Survivin does not contain intrinsic kinase activity we sought to determine what kinase(s) could be involved in stabilization or increase in phosphoTyr-15-Cdc2 by Survivin. Since Survivin inhibits caspase 3 activity and the Cdc2-targeting kinase Wee1 is a degradation target of active caspase 3, we performed immunoblotting assays on Survivin wt and T34A-transduced BaF3 cells and showed that cells over-expressing wt Survivin contain 2-fold higher levels of Wee1 protein as compared to those expressing vector or DN-Survivin. In addition, cells overexpressing wt Survivin contain >2-fold higher intact PARP protein levels which is another target of active caspase 3, as compared to cells transduced with MIEG vector. In order to test whether mimicking wt Survivin inhibition of caspase 3 could lead to increased Cdc2-Tyr15 phosphorylation, we evaluated the specific caspase 3 inhibitor Ac-DEVD-CHO. Inhibition of caspase 3 in BaF3 cells produced an increase in Cdc2Tyr15 phosphorylation in a dose-dependent manner, with 25 mM giving >4-fold higher intact PARP and >6-fold higher phospho-Tyr15 protein levels. Taken together, these results suggest that the antiapoptotic effect of Survivin is mediated through inactivation of Cdc2 by phosphorylation on Tyr-15 as a consequence of protection of the Wee1 kinase from degradation by caspase 3.

A Key Role for Poly(ADP-Ribose) Polymerase-1 Activity during Human Dendritic Cell Maturation

Poly(ADP-ribose) (PAR) polymerase (PARP)-1 is a nuclear enzyme regulating protein that functions by targeting PAR chains. Besides its classic role in DNA repair, PARP-1 is emerging as a key transcriptional regulator in different cell types including the immune ones. In this study, we investigated the role of PARP-1 in human dendritic cell (DC) function. We report that both PARP-1 mRNA and protein levels significantly increased during in vitro DC differentiation from monocytes. Of note, inhibitors of PARP-1 such as phenanthridinone and thieno[2,3-c]isoquinolin-5-one reduced expression of CD86 and CD83 in a concentration-dependent manner, having no effects on expression of CD80 and HLA-DR in mature DCs. In the same cultures, PARP-1 inhibitors also reduced production of IL-12 and IL-10. Addition of exogenous IL-12 to the culture medium partially restored CD86 expression in DCs exposed to PARP-1 inhibitors. In line with the role of PAR formation in NF-kappaB-dependent transactivation, we also report that phenanthridinone and thieno[2,3-c]isoquinolin-5-one impaired NF-kappaB and AP-1 subunit DNA binding activity in cellular extract of activated DCs. Finally, we show that PARP-1 inhibitors reduced the T cell allostimulatory activity of mature DCs, and that this reduction was prevented when DCs matured in the presence of PARP-1 inhibitors plus IL-12. Of note, nonproliferating T cells exposed to PARP-1 inhibitor-challenged DCs could undergo efficient proliferation when exposed to a subsequent activation stimulus such as anti-CD3 plus anti-CD-28. Together, data provide evidence for a key role of PARP-1 and poly ADP-ribosylation in DC immunocompetence and underscore the relevance of PARP-1 inhibitors to treatment of immune disorders.

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.

Preclinical evaluation of the dual specific Src/Abl kinase inhibitor AZD0530 in lung cancer

13108 Background: AZD0530 is a highly selective, orally available, dual specific Src/Abl kinase inhibitor in clinical development. We tested this agent in multiple lung cancer cell lines in vitro. We hypothesized that activity of AZD0530 may depend on the level of activated (pY416-)Src, and that Src inhibition may decrease Bcl-xL protein levels and lower the barrier to apoptosis. Methods: NSCLC (A549, Calu-1, Calu-6) and SCLC (H69, H526) cells were incubated with 0.001–100 μM AZD0530 for 1–72hrs. Proliferation (MTT), DNA-content (flow cytometry), and protein levels of Src, pY416-Src, PARP, and Bcl-xL (Western blotting) were assessed. Results: Basal pY416-Src was detectable in most cell lines except H526. AZD0530 decreased pY416-Src levels at submicromolar concentrations in pY416-Src positive cells. In A549, Calu-1 and Calu-6, AZD0530 blocked cell growth in a time- and dose-dependent way (IC50 = 7–25 μM) by arrest in G1, retaining a cytostatic effect at submicromolar concentrations in A549 and Calu-1. AZD0530 induced apoptosis in 10–22% of these cells at micromolar concentrations, accompanied by a decrease of Bcl-xL protein in A549 and Calu-1. In H69 and H526, growth inhibition by AZD0530 was limited (IC50 >100 μM). Conclusions: 1) AZD0530 induced apoptosis at micromolar concentrations, and inhibited cell growth at micromolar to submicromolar concentrations in some cell lines. 2) pY416-Src is a potential marker for drug responsiveness, but other factors should also be tested. 3) Decrease of Bcl-xL by AZD0530 may render cancer cells more sensitive to chemotherapy. These data suggest that Src kinase inhibitors merit further testing in lung cancer, both alone and in combination with other agents. (Support: Swiss National Science Foundation, Swiss Cancer League, AstraZeneca). [Table: see text]

Transgenic expression of human FGF-1 protects against hypoxic–ischemic injury in perinatal brain by intervening at caspase-XIAP signaling cascades

Perinatal hypoxia-ischemia (HI) is a major cause of neurological disability and mortality in infant and children. In the present study, we explored the neuroprotective efficacy of FGF-1 in a rat model of perinatal HI. Carotid ligation combined with hypoxia caused marked infarctions in the ipsilateral cerebral hemisphere with significant loss of ipsilateral striatal, cortical and hippocampal volumes. Morphological analyses revealed both apoptotic and necrotic form of neuronal death determined by Nissl histology, dark-field microscopy and TUNEL staining. HI induced a marked increase in activated caspase-9, caspase-3 and PARP cleavage at 12 h to 7 days after HI in brain areas displaying TUNEL (+) cells. In addition, expression of the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP) was decreased under similar conditions of HI. Expression of human FGF-1 in brain significantly reduced the extent of both apoptotic and necrotic injury caused by HI. FGF-1 attenuated the HI-induced increase in activated caspase-3, caspase-9 and cleaved PARP protein levels and markedly blocked the HI-induced decrease in XIAP expression under the conditions at which FGF-1 showed significant neuroprotection. These findings demonstrate that FGF-1 prevents the onset of both apoptotic and necrotic death in neurons otherwise "destined to die" following hypoxic-ischemic injury by intervening at the level of caspase-signaling cascades and by restoring prosurvival protein XIAP expression in central neurons.

Decreased PARP and procaspase-2 protein levels are associated with cellular drug resistance in childhood acute lymphoblastic leukemia

AbstractDrug resistance in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) is associated with impaired ability to induce apoptosis. To elucidate causes of apoptotic defects, we studied the protein expression of Apaf-1, procaspases-2, -3, -6, -7, -8, -10, and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) in cells from children with acute lymphoblastic leukemia (ALL; n = 43) and acute myeloid leukemia (AML; n = 10). PARP expression was present in all B-lineage samples, but absent in 4 of 15 T-lineage ALL samples and 3 of 10 AML cases, which was not caused by genomic deletions. PARP expression was a median 7-fold lower in T-lineage ALL (P < .001) and 10-fold lower in AML (P < .001) compared with B-lineage ALL. PARP expression was 4-fold lower in prednisolone, vincristine and L-asparaginase (PVA)-resistant compared with PVA-sensitive ALL patients (P < .001). Procaspase-2 expression was 3-fold lower in T-lineage ALL (P = .022) and AML (P = .014) compared with B-lineage ALL. In addition, procaspase-2 expression was 2-fold lower in PVA-resistant compared to PVA-sensitive ALL patients (P = .042). No relation between apoptotic protease-activating factor 1 (Apaf-1), procaspases-3, -6, -7, -8, -10, and drug resistance was found. In conclusion, low baseline expression of PARP and procaspase-2 is related to cellular drug resistance in childhood acute lymphoblastic leukemia. (Blood. 2005;106:1817-1823)

Phenylbutyrate-induced apoptosis is associated with inactivation of NF-kappaB IN HT-29 colon cancer cells.

Cytotoxic chemotherapy has been used to treat patients with metastatic colorectal cancer with limited success. Therefore novel chemotherapeutic approaches are needed. Based on encouraging preclinical data, there has been an interest in developing derivatives of butyrate as clinically applicable agents. The purpose of this study was to investigate the effects of phenylbutyrate (PB), a butyrate analogue, on the cell growth and apoptosis in a colon cancer cell model. Growth curves, flow cytometric studies, Western blotting, DNA binding assays and transient transfection experiments were performed in vitro using the colon cancer cell line HT-29 after exposure to PB. Exposure of HT-29 colon cancer cells to PB resulted in growth inhibition and induction of apoptosis as measured by annexin V staining. This increase in apoptosis was associated with a decrease in mitochondrial membrane potential, an increase in caspase-3 activity and a decrease in intact PARP protein levels. Since NF-kappaB plays a pivotal role in the regulation of apoptosis, we explored the effects of PB on the DNA binding and transcriptional activity of this transcription factor. After PB treatment, NF-kappaB-DNA binding was markedly decreased and specifically, this decreased DNA binding was observed in the p50:p65 heterodimer. The decreased NF-kappaB DNA binding was observed as early as 3 h after PB treatment, while no apparent changes in annexin V binding were detected until 12 h after PB treatment. Untreated HT-29 cells transfected with a kappaB-luciferase reporter plasmid demonstrated significant constitutive activity of the kappaB binding site, which was markedly decreased after treating the cells with PB. These results suggest that PB-induced apoptosis may be partly regulated through the inactivation of NF-kappaB. PB, an oral butyrate analogue, may have therapeutic potential in colon cancer.

A nuclear factor other than Sp1 binds the GC-rich promoter of the gene encoding rat poly(ADP-ribose) polymerase in vitro

Poly(ADP-ribose) polymerase is a nuclear enzyme that has been shown to exert a key role in many important cellular functions, including DNA repair. Its activity was shown to vary substantially between tissues; the testis and the thymus expressed the highest levels of PARP whereas the liver and the kidney (as well as a few other tissues) expressed only low levels of PARP proteins in vivo. The GC-rich nature of its upstream gene promoter, along with the lack of TATA and CAAT boxes, a feature common to most housekeeping genes, is consistent with a major regulatory function played by the positive transcription factor Sp1 in rat PARP gene transcription. Sp1 was indeed recently shown to interact with five distinct GC or GT boxes present in the rat PARP promoter. However, the observation that PARP activity was lower in rat liver than in other tissues was shown not to be the result of reduced Sp1 activity in liver cells but rather suggests the interplay of nuclear proteins other than Sp1 that are required to restrict PARP expression in this organ and maybe in others (such as the kidney). In this study, we investigated this possibility further by defining whether other nuclear proteins might bind the PARP promoter to modulate its transcription in liver cells. As a result, we identified a nuclear factor distinct from Sp1 that binds the PARP promoter at a site overlapping the F2 Sp1 element previously identified. Our results suggest that this protein likely belongs to the CTF-NF1 family of transcription factors.

A nuclear factor other than Sp1 binds the GC-rich promoter of the gene encoding rat poly(ADP-ribose) polymerase in vitro

Poly(ADP-ribose) polymerase is a nuclear enzyme that has been shown to exert a key role in many important cellular functions, including DNA repair. Its activity was shown to vary substantially between tissues; the testis and the thymus expressed the highest levels of PARP whereas the liver and the kidney (as well as a few other tissues) expressed only low levels of PARP proteins in vivo. The GC-rich nature of its upstream gene promoter, along with the lack of TATA and CAAT boxes, a feature common to most housekeeping genes, is consistent with a major regulatory function played by the positive transcription factor Sp1 in rat PARP gene transcription. Sp1 was indeed recently shown to interact with five distinct GC or GT boxes present in the rat PARP promoter. However, the observation that PARP activity was lower in rat liver than in other tissues was shown not to be the result of reduced Sp1 activity in liver cells but rather suggests the interplay of nuclear proteins other than Sp1 that are required to restrict PARP expression in this organ and maybe in others (such as the kidney). In this study, we investigated this possibility further by defining whether other nuclear proteins might bind the PARP promoter to modulate its transcription in liver cells. As a result, we identified a nuclear factor distinct from Sp1 that binds the PARP promoter at a site overlapping the F2 Sp1 element previously identified. Our results suggest that this protein likely belongs to the CTF-NF1 family of transcription factors.