Physical and functional interaction of the TPL2 kinase with nucleophosmin.

Tumor Progression Locus 2 (TPL2) is widely recognized as a cytoplasmic mitogen-activated protein 3 kinase with a prominent role in the regulation of inflammatory and oncogenic signal transduction. Herein we report that TPL2 may also operate in the nucleus as a physical and functional partner of nucleophosmin (NPM/B23), a major nucleolar phosphoprotein with diverse cellular activities linked to malignancy. We demonstrate that TPL2 mediates the phosphorylation of a fraction of NPM at threonine 199, an event required for its proteasomal degradation and maintenance of steady-state NPM levels. Upon exposure to ultraviolet C, Tpl2 is required for the translocation of de-phosphorylated NPM from the nucleolus to the nucleoplasm. NPM is an endogenous inhibitor of HDM2:p53 interaction and knockdown of TPL2 was found to result in reduced binding of NPM to HDM2, with concomitant defects in p53 accumulation following genotoxic or ribosomal stress. These findings expand our understanding of the function of TPL2 as a negative regulator of carcinogenesis by defining a nuclear role for this kinase in the topological sequestration of NPM, linking p53 signaling to the generation of threonine 199-phosphorylated NPM.

Chemotherapy and radiotherapy downregulate the activity and expression of DNA methyltransferase and enhance Bcl-2/E1B-19-kDa interacting protein-3-induced apoptosis in human colorectal cancer cells.

Bcl-2/E1B 19-kDa interacting protein 3 (BNIP3) is a proapoptotic protein whose expression level is often low in colorectal cancer (CRC) cells due to the BNIP3 gene promoter DNA methylation by DNA methyltransferase (DNMT). It is known that chemotherapy and radiotherapy suppress CRC through inducing tumor apoptosis. However, the molecular mechanisms underlying chemotherapy and radiotherapy-induced apoptosis of CRC cells are not well defined. In this study, we observed that the expression level of BNIP3 in colon cancer cells was significantly increased by treatment with therapeutic agents and radiation in vitro. The BNIP3 protein level in CRC tissues from patients who received preoperative concurrent chemotherapy was significantly higher than in those who received surgery alone. Furthermore, treatment with chemotherapeutic agents and radiation significantly decreased the DNMT1 expression level and enzymatic activity. Both expression level and activity of DNMT1 were inversely correlated with the expression level of BNIP3 in colon carcinoma cells after treatment with chemotherapeutic agents and radiation. Consistent with increased BNIP3 expression, chemotherapeutic agents and radiation induced colon carcinoma cell apoptosis in a dose-dependent manner. Based on these observations, we conclude that chemotherapy and radiotherapy inhibit DNMT1 expression to upregulate BNIP3 expression to promote CRC cell apoptosis. And, BNIP3 may play a role in the caspase-dependent apoptosis pathways, mainly during treatment with chemotherapy and radiotherapy.

Slug inhibition upregulates radiation-induced PUMA activity leading to apoptosis in cholangiocarcinomas.

Resistance of cholangiocarcinoma to irradiation therapy is a major problem in cancer treatment. Slug, a snail family transcription factor, is a suppressor of PUMA (p53 upregulated modulator of apoptosis), which has been shown to be involved in the control of apoptosis. In this study, we investigated whether the modulation of Slug expression, using adeno-associated-virus-mediated transfer of siRNA targeting Slug gene (rAAV2-Slug siRNA), affects cholangiocarcinoma sensitivity to radiation. In the present study, we used rAAV2-Slug siRNA to downregulate the expression of Slug in QBC939 cholangiocarcinoma cell lines in vitro before γ-irradiation. In vivo studies were done with orthotopic cholangiocarcinoma, and radiosensitivity was evaluated both in vitro and in vivo. rAAV2-Slug siRNA transfection resulted in downregulation of the levels of Slug in QBC939 cells. In addition, rAAV2-Slug siRNA, in combination with radiation, increased levels of the PUMA, which contributes to the radiosensitivity of cholangiocarcinomas. Finally, treatment with rAAV2-Slug siRNA plus γ-irradiation completely regressed tumor growth in orthotopic cholangiocarcinomas model. In summary, integrating gene therapy with radiotherapy could have a synergistic effect, thereby improving the survival of patients with cholangiocarcinomas.

[Analysis of PARP10 tissue expression profile, interactive protein and UV stress reaction].

One pair of primers were designed and synthesized based on the cDNA sequence encoding Homo sapiens poly (ADP-ribose) polymerase family, member 10 (PARP10) reported on the GenBank. The cDNA sequence encoding PARP10 was cloned from 293FT cell by RT-PCR. Then the RT-PCR product was cloned into pCMV-Myc and pEGFP-C1 plasmids. The interaction between PARP10 and beta-actin was identified through immuno-precipitation and laser confocal microscopy. Extensive expression of PARP10 in mouse tissues was confirmed by RT-PCR. Besides, Western blotting analysis indicated that cell injury caused by UV treatment could promote the expression of PARP10. The results in this paper would benefit further study of PARP10.

p53 Binding to the p21 promoter is dependent on the nature of DNA damage.

p53 Is a tumor suppressor that integrates signals from different stress induced signalling pathways, regulates cell cycle arrest, senescence, apoptosis and DNA repair. How p53 dictates cell fate is unclear. As a major transcriptional target of p53 in response to cellular stress, p21 is a key component in cell cycle control and apoptosis, directing an anti-apoptotic response following DNA damage. It is therefore likely that p53-dependent regulation of p21 contributes, at least in part, how p53 influences cellular outcome upon DNA damage. Here we compare the p53-dependent transcriptional regulation of p21 in response to DNA damage by ultraviolet (UV) radiation and ionizing radiation (IR). We demonstrate that despite comparable levels of p53 accumulation by both types of DNA damage, IR causes significant, early accumulation of p21 not seen in UV-damaged cells, with a substantially different cell cycle profile. Whereas UV and IR both target p21 protein for degradation immediately after DNA damage, differential post-damage p21 transcription is accountable for the disparity in p21 protein levels. Chromatin immunoprecipitation studies reveal that p53 displays a clear bias against the p21 promoter in UV-damaged cells compared to IR-damaged cells. We note differential post-translational modifications of nuclear p53 between UV and IR treatment. Furthermore we demonstrate that this disparity correlated with reduced histone acetylation on the TATA box within the p21 promoter following UV treatment. This suggests that the nature of DNA damage enables p53 to selectively discriminate between promoters in the induction of target genes, thereby regulating their expression and subsequent cellular outcome.

Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress.

Following genotoxic stress, p53 either rescues a damaged cell or promotes its elimination. The parameters determining a specific outcome of the p53 response are largely unknown. In mouse fibroblasts treated with different irradiation schemes, we monitored transcriptional and non-transcriptional p53 activities and identified determinants that initiate an anti- or a pro-apoptotic p53 response within the context of p53-independent stress signaling. The primary, transcription-mediated p53 response in these cells is anti-apoptotic, while induction of p53-dependent apoptosis requires an additional, transcription-independent p53 activity, provided by high intracellular levels of activated p53. High intracellular levels of p53 were selectively generated after apoptosis-inducing high-dose UV-irradiation, and correlated with a strongly delayed upregulation of Mdm2. Following high-dose UV-irradiation, p53 accumulated in the cytoplasm and led to activation of the pro-apoptotic protein Bax. As p53-dependent Bax-activation is transcription-independent, we postulated that certain transcription-deficient mutant p53 proteins might also exert this activity. Indeed we found an endogenous, transcription-inactive mutant p53 that upon genotoxic stress induced Bax-activation in vivo. Our results demonstrate the impact and in vivo relevance of non-transcriptional mechanisms for wild-type and mutant p53-mediated apoptosis.

Dark pulse suppression of P-ERK and c-Fos in the hamster suprachiasmatic nuclei.

It is well-established that light pulses regulate components of the extracellular signal-regulated kinases I/II (ERK) cascade in the suprachiasmatic nuclei (SCN) circadian clock. These events are important for photic-resetting of the circadian clock. The SCN circadian clock is also reset by pulses of dark, but it is unknown if this stimulus alters the activity of ERK, the transcription factor Elk-1 or expression of the immediate early gene c-fos in the SCN. Using Syrian hamsters free-running in constant light, we determined the effects of dark pulses on these factors in the SCN. In constant light, levels of phosphorylated ERK (P-ERK) showed significant circadian variation in the Syrian hamster SCN, while levels of c-Fos or phosphorylated Elk-1 (P-Elk-1) did not. A 6-h dark pulse beginning at circadian time (CT) 8 down-regulated expression of P-ERK and c-Fos, but not P-Elk-1, in the SCN. Following termination of the pulse, levels of c-Fos increased above time-matched control values, while P-ERK expression did not. When given at the beginning of the subjective night (CT13), a 6-h dark pulse did not phase-shift behavioural rhythms and failed to alter the expression of c-Fos, P-ERK, or P-Elk-1 in the SCN. At the level of the visual thalamus, expression of c-Fos in the intergeniculate leaflet was higher during the subjective night as compared to the subjective day, although dark pulses had no robust effects on expression of c-Fos or P-ELK-1 in this structure. We conclude that dark-pulse resetting of the circadian clock is complex and involves both non-photic and photic components.

Silibinin inhibits ultraviolet B radiation-induced mitogenic and survival signaling, and associated biological responses in SKH-1 mouse skin.

Ultraviolet B (UVB) radiation is a complete skin carcinogen causing DNA damage as a tumor-initiating event and activating signaling cascades that play a critical role in its tumor-promoting potential. Recently we reported that a naturally occurring flavonoid, silibinin, protects UVB-induced skin damages and prevents photocarcinogenesis. Here we examined silibinin efficacy on acute and chronic UVB-caused mitogen-activated protein kinases (MAPKs) and AKT activation and associated biological responses in SKH-1 hairless mouse skin. A single UVB exposure at 180 mJ/cm2 dose resulted in varying degrees of ERK1/2, JNK1/2, MAPK/p38 and AKT phosphorylation at various time-points in mouse skin; however, topical application of silibinin prior to or immediately after UVB exposure, or its dietary feeding strongly inhibited the activation of these molecules at all the time-points examined. Stronger effects of silibinin towards inhibition of UVB-caused phosphorylation of MAPKs and AKT were also observed in a chronic UVB (180 mJ/cm2/day for 5 days) exposure protocol. Immunohistochemical analysis of chronically exposed skin sections showed that silibinin treatment in all three protocols increases UVB-induced p53-positive cells and decreases UVB-caused cell proliferation, apoptotic and sunburn cells. These findings suggest that silibinin inhibits UVB-induced MAPK and AKT signaling and increases p53 in mouse skin, and that these effects of silibinin possibly lead to a decrease in UVB-caused proliferation and apoptosis, which might, in part, be responsible for its overall efficacy against photocarcinogenesis.

Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation.

Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.

Silibinin protects against photocarcinogenesis via modulation of cell cycle regulators, mitogen-activated protein kinases, and Akt signaling.

Here, we assessed the protective effect of silibinin on UVB-induced skin carcinogenesis in SKH-1 hairless mice. Topical application of silibinin before or immediately after UVB exposure or its dietary feeding resulted in a strong protection against photocarcinogenesis, in terms of tumor multiplicity (60-66%; P < 0.001), tumor volume per mouse (93-97%; P < 0.001) and tumor volume per tumor (80-91%; P < 0.001). Silibinin also moderately inhibited tumor incidence (5-15%; P < 0.01) and delayed tumor latency period (up to 4 weeks; P < 0.01-0.001). To investigate in vivo molecular mechanisms of silibinin efficacy, tumors and uninvolved skin from tumor-bearing mice were examined immunohistochemically for proliferation, p53, apoptosis, and activated caspase-3. Silibinin treatment showed a strong decrease (P < 0.001) in proliferating cell nuclear antigen-positive cells and an increase in p53-positive (P < 0.005-0.001), terminal deoxynucleotidyltransferase-mediated nick end labeling-positive (P < 0.005-0.001), and cleaved caspase-3-positive cells (P < 0.001). Western blot analysis of normal skin and tumor lysates showed that silibinin decreases the levels of cyclin-dependent kinase 2 and cyclin-dependent kinase 4 and associated cyclins A, E, and D1, together with an up-regulation of Cip1/p21, Kip1/p27, and p53. Silibinin also showed a strong phosphorylation of extracellular signal-regulated protein kinase 1/2, stress-activated protein kinase/c-JUN NH2-terminal kinase 1/2, and p38 mitogen-activated protein kinases but inhibited Akt phosphorylation and decreased survivin levels with an increase in cleaved caspase-3. Together, these results show a strong preventive efficacy of silibinin against photocarcinogenesis, which involves the inhibition of DNA synthesis, cell proliferation, and cell cycle progression and an induction of apoptosis. Furthermore, these results also identify in vivo molecular mechanisms of silibinin efficacy against photocarcinogenesis.

Phosphatidylinositide 3-kinase/AKT in radiation responses.

Ionizing or ultraviolet radiation-induced cellular survival signaling pathways induce development of cancer and insensitivity of tumor cells to radiation therapy. Accumulating evidence suggests that the phosphatidylinositide 3-kinase (PI3K)/AKT signal pathway is a major contributor to radioresistance. In many cell types PI3K/AKT signaling is a key cytoprotective response downstream of the EGFR family receptors and mediated carcinogenesis. Cytokines, such as HGF, IGF-I, and IL-6 also protects cells against apoptosis induced by radiation through PI3K/AKT pathway. The mechanics by which PI3K/AKT signaling functions in radiation responses may include its regulation of mitochondrial proteins, transcription factors, translation machinery, and cell-cycle progression. In addition, cross-talk between the PI3K/AKT pathway and mitogen-activated protein kinases, protein kinase A, and protein kinase C signal pathway may also play an important role.

Cancer predisposition in mice deficient for the metastasis-associated Mts1(S100A4) gene.

Metastasis-promoting Mts1(S100A4) protein belongs to the S100 family of Ca(2+)-binding proteins. A mouse strain with a germ-line inactivation of the S100A4 gene was generated. The mice were viable and did not display developmental abnormalities in the postnatal period. However, an abnormal sex ratio was observed in the litters with the S100A4-/- genotype, raising the possibility of a certain level of embryonic lethality in this strain. In all, 10% of 10-14-month-old S100A4-null animals developed tumors. This is a characteristic feature of mouse strains with inactivated tumor suppressor genes. Spontaneous tumors of S100A4-/- mice were p53 positive. Recently, we have shown that S100A4 interacts with p53 tumor suppressor protein and induces apoptosis. We proposed that impairment of this interaction could affect the apoptosis-promoting function of p53 that is involved in its tumor suppressor activity. The frequency of apoptosis in the spleen of S100A4-/- animals after whole-body gamma-irradiation was reduced compared to the wild-type animals. The same was true for the transcriptional activation of the p53 target genes - waf/p21/cip1 and bax. Taken together, these observations indicate that spontaneous tumors in S100A4-/- mice are a result of functional destabilization of p53 tumor suppressor gene.

p53 binding to target sites is dynamically regulated before and after ionizing radiation-mediated DNA damage.

Although radiation therapy has been an important modality for cancer treatment, the molecular mechanisms underlying the overall genomic response of mammalian cells to radiation are not well characterized. The success of radiation therapy using ionizing radiation relies upon the regulation of both the cell cycle and apoptosis, as conferred by the activation of DNA damage-responsive genes. To better understand the key players involved in this response, expression-profiling experiments were performed using custom-made cDNA microarrays. In MOLT-4 lymphoma tumor cells, the induction of target gene products following irradiation supports a major role for p53 as a transcriptional activator, but also invokes questions regarding conditional transcription regulation following irradiation. Using chromatin immunoprecipitation (ChIP), p53 binding to chromatin was examined following irradiation using primers that are specific for p53 binding sites in target genes. PCR analysis indicates dynamic target gene binding. Thus, at 8 hours following radiation treatment, the p21 and puma promoter sites were characterized by relative increases in chromatin precipitation, while the bax promoter site was not. Because the binding of p53 to these sites only changed modestly following radiation, other studies were conducted to characterize the presence of constitutive binding to putative p53 DNA binding sites in several other genes.

Dynamics of the p53-Mdm2 feedback loop in individual cells.

The tumor suppressor p53, one of the most intensely investigated proteins, is usually studied by experiments that are averaged over cell populations, potentially masking the dynamic behavior in individual cells. We present a system for following, in individual living cells, the dynamics of p53 and its negative regulator Mdm2 (refs. 1,4-7): this system uses functional p53-CFP and Mdm2-YFP fusion proteins and time-lapse fluorescence microscopy. We found that p53 was expressed in a series of discrete pulses after DNA damage. Genetically identical cells had different numbers of pulses: zero, one, two or more. The mean height and duration of each pulse were fixed and did not depend on the amount of DNA damage. The mean number of pulses, however, increased with DNA damage. This approach can be used to study other signaling systems and suggests that the p53-Mdm2 feedback loop generates a 'digital' clock that releases well-timed quanta of p53 until damage is repaired or the cell dies.

Radiation-induced apoptosis in scid mice spleen after low dose irradiation.

To assess the radioadaptive response of the whole body system in mice, we examined the temporal effect of low dose priming as an indicator of challenging irradiation-induced apoptosis through a p53 tumor suppressor protein- mediated signal transduction pathway. The p53 protein also plays an important role both in cell cycle control and DNA repair through cellular signal transduction. Using severe combined immunodeficiency mice defective in DNA-dependent protein kinase catalytic subunit, we examined the role of DNA-dependent protein kinase activity in radioadaptation induced by low dose irradiation. Specific pathogen free 5-week-old female severe combined immunodeficiency mice and the parental mice (CB- 17 Icr +/+) were irradiated with X-ray at 3.0 Gy at 1, 2, 3 or 4 weeks after the conditioning irradiation at 0.15, 0.30, 0.45 or 0.60 Gy. The mice spleens were fixed for immunohistochemistry 12 h after the challenging irradiation. The p53-dependent apoptosis related Bax proteins on formalin-fixed paraffin-embedded sections were stained by the avidin-biotin peroxidase complex method. The apoptosis incidence in the sections was measured by hematoxylin-eosin staining. The frequency of Bax- and apoptosis-positive cells increased up to 12 h after the challenging irradiation in the spleen of both mice. However, these cells were not observed after a low dose irradiation at 0.15-0.60 Gy. When pre-irradiation at 0.45 Gy 2 weeks before the challenging irradiation at 3.0 Gy was performed, Bax accumulation and apoptosis induced by challenging irradiation were depressed in the spleens of CB-17 Icr +/+ mice, but not in severe combined immunodeficiency mice. These data suggest that DNA-dependent protein kinase might play a major role in radioadaptation induced by pre-irradiation with a low dose in mice spleen. We expect that the present findings will provide useful information in the health care of space crews.

Nitric oxide promotes p53 nuclear retention and sensitizes neuroblastoma cells to apoptosis by ionizing radiation.

Nitric oxide (NO) is a potent activator of the p53 tumor suppressor protein. However, the mechanisms underlying p53 activation by NO have not been fully elucidated. We previously reported that a rapid downregulation of Mdm2 by NO may contribute to the early phase of p53 activation. Here we show that NO promotes p53 nuclear retention and inhibits Mdm2-mediated p53 nuclear export. NO induces phosphorylation of p53 on serine 15, which does not require ATM but rather appears to depend on the ATM-related ATR kinase. An ATR-kinase dead mutant or caffeine, which blocks the kinase activity of ATR, effectively abolishes the ability of NO to cause p53 nuclear retention, concomitant with its inhibition of p53 serine 15 phosphorylation. Of note, NO enhances markedly the ability of low-dose ionizing radiation to elicit apoptotic killing of neuroblastoma cells expressing cytoplasmic wild-type p53. These findings imply that, through augmenting p53 nuclear retention, NO can sensitize tumor cells to p53-dependent apoptosis. Thus, NO donors may potentially increase the efficacy of radiotherapy for treatment of certain types of cancer.

Didox (a novel ribonucleotide reductase inhibitor) overcomes Bcl-2 mediated radiation resistance in prostate cancer cell line PC-3.

In this study, we investigated the influence of Bcl-2 overexpression on the radiosensitizing potential of Didox (DX; 3,4-Dihydroxybenzohydroxamic acid), a novel ribonucleotide reductase inhibitor, in p53-null prostate cancer cell line PC-3. The PC-3 cells were transfected with vector alone or ectopically overexpressed with CMV-Bcl-2 construct. The effect of radiation (IR) or DX alone and in combination (pre and post IR exposure of DX) on cell survival was determined by colony-forming assay. The impact of these two treatments on the cell cycle was determined by flow cytometry. To further understand the molecular mechanism of DX-mediated radiosensitization, induction of pro-survival and pro-apoptotic factors were determined by Western blot and gel-shift assays respectively. When compared to PC-3/Bcl-2 cells (SF(2)=0.84; D(0)=437cGy), the PC-3/vector cells (SF(2)=0.4; D(0)=235cGy) were significantly sensitive to ionizing radiation (p<0.001). Exposure of DX at 5 microM concentration prior or post to radiation in both PC-3/vector and PC-3/Bcl-2 transfectants caused an increase in radiation enhancement ratios. A significant reduction in G(2)M phase was observed in cells exposed to DX post IR when compared to cells exposed to IR alone. Exposure to DX after radiation in PC-3/vector significantly abrogated radiation-induced Bcl-2 upregulation, with a concomitant induction of bax protein. In PC-3/Bcl-2 transfectants, DX exposure after IR caused an induction of bax protein. Gel shift assays indicated that in PC-3/vector cells when exposed to IR caused an induction of NFkappa-B activity however, DX down regulated the NFkappa-B activity. Radiation-induced NFkappa-B activity was abrogated in pre and post DX exposure in combination with IR. These findings indicate that DX mediates a potent radiosensitizing effect in p53 null prostate cancer cells by overcoming radiation induced NFkappa-B activity and Bcl-2 expression.

Lymphocytes treated by extracorporeal photopheresis demonstrate a drop in the Bcl-2/Bax ratio: a possible mechanism involved in extracorporeal-photopheresis-induced apoptosis.

BACKGROUND: Recently, apoptosis has been identified in treated lymphocytes, prior to their re-infusion, when tested ex vivo. Previous work has demonstrated a close association between the genes p53, Bcl-2 and Bax and apoptosis induced by UV irradiation. OBJECTIVES: We wanted to establish whether the expression of the protein product of these genes was altered in lymphocytes treated with extracorporeal photopheresis (ECP) prior to re-infusion and therefore possibly implicated in the early apoptosis observed. METHOD: Lymphocytes were isolated immediately before treatment and immediately prior to re-infusion and tested for intracellular levels of p53, Bcl-2 and Bax proteins. RESULTS: No increase in p53 expression was observed at re-infusion; however, the mean fluorescent intensity ratio of the apoptotic inhibitor protein Bcl-2 to the apoptosis-inducing protein Bax dropped significantly. CONCLUSION: The early apoptosis observed in ECP-treated lymphocytes at re-infusion might be attributed to dysregulation in the expression of the apoptotic genes Bcl-2 and Bax.

Differential post-transcriptional regulation of p21WAF1/Cip1 levels in the developing nervous system following gamma-irradiation.

Radiation-induced death in the developing brain is p53-dependent. However, genetic studies indicate that the signalling pathways that couple irradiation to p53 expression can vary between different developing neural populations [Herzog et al. (1998) Science, 280, 1089-1091]. Here we establish that signalling downstream of p53 also exhibits brain region-specific differences that are associated with the relative vulnerability of some cell populations to radiation-induced killing in the mouse. Following gamma-irradiation, p53 and p21WAF1/cip1, but not Bax, protein levels increased in the developing cerebellum. In contrast, neither p21WAF1/cip1 nor Bax protein levels were elevated in the retina following irradiation, despite increased p53 expression. In the retina, p53 expression was associated with cells destined to die, whereas in the cerebellum, p53 was expressed in both radiation-sensitive and radiation-resistant neuroblasts of the external granule cell layer. Although p21WAF1/cip1 mRNA was expressed in all p53-positive neuroblasts after irradiation, p21WAF1/cip1 protein was only detected in radiation-resistant neuroblasts of the cerebellum. Thus, p21WAF1/cip1 was subject to post-transcriptional regulation with p21WAF1/cip1 protein only accumulating in cells destined to survive irradiation. Nevertheless, p21WAF1/cip1 function was not essential for radiation resistance, as postmitotic neuroblasts in the external granule cell layer were spared in p21WAF1/cip1 knockout mice.

Defects in transcription coupled repair interfere with expression of p90(MDM2) in response to ultraviolet light.

Ultraviolet (UV) irradiation transiently stabilizes p53 through a mechanism that may require a decrease in the activity of the ubiquitin ligase, p90(MDM2). Conversely, the recovery of low levels of p53 following UV exposure may depend on an increase in p90(MDM2). The level of p90(MDM2) is increased by UV light following the p53-dependent induction of an internal mdm2 promoter, P2. If this induction of mdm2 were critical for the recovery of low levels of p53 following UV exposure, defects in mdm2's transcription would result in a prolonged increase in p53. Cells defective in transcription coupled repair (TCR) maintain high levels of p53 for a prolonged period following UV exposure. Such cells also have defects in general transcription after UV irradiation. We investigated whether TCR-deficient cells express diminished levels of mdm2 mRNA and p90(MDM2) following UV exposure. We found that transcription of mdm2 was reduced in TCR-deficient cells. The uninducible mdm2 promoter, P1, was more sensitive to the inhibitory effects of UV irradiation than the P2 promoter. The decrease in transcription from the P1 promoter was sufficient to reduce the level of p90(MDM2) and correlated with a prolonged increase in p53. Thus, p53-independent transcription of mdm2 appears critical to p53's regulation.