Growth arrest and DNA damage-45 alpha (GADD45alpha)

Regulation of cell cycle and growth is integral for cell survival. The intricate mechanisms that control proliferation and cell cycle are numerous. The growth arrest and DNA damage (GADD)-inducible gene family is often up-regulated in response to various environmental stresses and drug therapies. GADD45alpha was the first stress-inducible gene determined to be up-regulated by p53 and is also a target for the p53 homologues, p63 and p73. When GADD45alpha is deleted or repressed, cells show uncontrolled proliferation. Furthermore, decreased GADD45alpha expression is also considered a survival mechanism, as cancer cells without this control can evade the apoptotic pathway leading to increased tumourigenesis. Drug therapies can act to directly or indirectly up-regulate GADD45alpha and promote apoptosis. As GADD45alpha is an essential component of many metabolic pathways that control proliferating cancer cells, it presents itself as an emerging drug target worthy of further investigation.     

A polymorphism but no mutations in the GADD45 gene in breast cancers

The p53 gene product is part of a pathway regulating growth arrest at the G₁ checkpoint of the cell cycle. Mutation of other components of this pathway, including the products of the ataxia telangiectasia (AT), GADD45, mdm2, and p21^WAF1/CIP1 genes may have effects comparable to mutations in the p53 gene. The GADD45 gene is induced by ionizing radiation and several DNA-damaging xenobiotics. Induction requires the binding of wild-type p53 to an evoulutionarily highly conserved putative intronic p53 binding site in intron 3 of GADD45. We recently analyzed the entire coding region of the p53 gene in primary breast cancers of Midwestern white women and found 21 mutations among 53 tumors (39,6%). We now have shown by direct sequencing that there are no mutations in the intronic p53 binding site of the GADD45 gene in any of the 53 primary breast cancers and no mutations in the entire coding region of the GADD45 gene in a subset of 26 consecutive tumors (12 with p53 mutation and 14 without p53 mutation). The only sequence variation detected was a common polymorphism in intron 3. The absence of mutations in the GADD45 gene, including the putative p53-binding intronic site, suggests that this gene is not a frequent target of mutations in breast cancer. Although mutations of the p53 gene have been studied in a wide spectrum of human cancers, GADD45 has not been examined in any tumor or cell line to the best of our knowledge. Our results raise the possibility that mutation of the GADD45 gene alone is not functionally equivalent to loss of wild-type p53 activity. Received: 14 September 1995     

Human melanoma cell line UV responses show independency of p53 function.

UV radiation-induced mutation of the p53 gene is suggested as a causative event in skin cancer, including melanoma. We have analyzed here p53 mutations in melanoma cell lines and studied its stabilization, DNA-binding activity, and target gene activation by UVC. p53 was mutated in three of seven melanoma cell lines. However, high levels of p53 were detected in all cell lines, including melanoma cells with wild-type p53, with the exception of one line with a truncated form. Upon UV induction, p53 accumulated in lines with wild-type p53, and p53 target genes p21Cip1/Waf1, GADD45, and mdm2 were induced, but the induction of p21Cip1/Waf1 was significantly delayed as compared with the increase in p53 DNA-binding activity. However, despite p53 target gene induction, p53 DNA-binding activity was absent in one melanoma line with wild-type p53, and p53 target genes were induced also in cells with mutant p53. In response to UV, DNA replication ceased in all cell lines, and apoptosis ensued in four lines independently of p53 but correlated with high induction of GADD45. The results suggest that in melanoma, several p53 regulatory steps are dislodged; its basal expression is high, its activation in response to UV damage is diminished, and the regulation of its target genes p21Cip1/Waf1 and GADD45 are dissociated from p53 regulation.     

Induction of p53-dependent activation of the human proliferating cell nuclear antigen gene in chromatin by ionizing radiation

A human fibroblast cell line with conditional p53 expression displayed a p53-dependent increase in both the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) after exposure to ionizing radiation (IR). The combination of p53 induction and IR cooperated to activate a transiently expressed human PCNA promoter-reporter gene via a p53-responsive element. Chromatin immunoprecipitation assays with antibodies specific for p53 or p300/CREB-binding protein revealed specific p53-dependent enrichment of PCNA promoter sequences in immunoprecipitates of sheared chromatin prepared from irradiated cells. Maximal and specific association of acetylated histone H4 with the PCNA promoter also depended on p53 induction and exposure to IR. These data demonstrate p53 binding to a target site in the PCNA promoter, recruitment of p300/CREB-binding protein, and localized acetylation of histone H4 in an IR-dependent manner. These molecular events are likely to play a role in mediating activation of PCNA gene expression by p53 during the cellular response to DNA damage. The analyses indicate that the combination of p53 induction and IR activate the PCNA gene via mechanisms similar to that of p21/wild-type p53-activated factor but to a lesser extent. This differential regulation of PCNA and p21/wild-type p53-activated factor may establish the proper ratio of the two proteins to coordinate DNA repair with cell cycle arrest.     

p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia

Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.     

Blockage of NF-κB Induces Serine 15 Phosphorylation of Mutant p53 by JNK Kinase in Prostate Cancer Cells

The p53 tumor suppressor gene plays an important role during induction of apoptosis in cancer. In contrast, NF-κB prevents apoptosis in response to chemotherapeutic agents and is a critical regulator of cell survival. Despite the riches of information on the regulation of wild-type p53 function by phosphorylation, nothing is known about the modulation of mutant p53 activity by phosphorylation. Here we report that inhibition of NF-κB in DU145 prostate cancer cells results in p53 mutant phosphorylation at serine 15 (Ser15), leading to an increase of p53 stability, DNA binding and gain of function. Serine 15-phosphorylation is due to GADD45a-dependent induction of JNK kinase, which can be blocked by SP600125, a JNK kinase inhibitor. Furthermore, inhibition of GADD45a by small interfering RNA blocks JNK activation and abrogates Ser15 phosphorylation. Together, these results highlight the importance of Ser15 phosphorylation in regulating the oncogenic function of mutant p53 and apoptosis induction in the context of the NF-κB/IκB signaling pathway.