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Biomedical subjects

M Ljungman

Publications and source records attributed to M Ljungman.

At least 19 recordsLinked to original sources
PubMed2001

P53 plays a protective role against UV- and cisplatin-induced apoptosis in transcription-coupled repair proficient fibroblasts.

We previously reported that transcription-coupled repair (TCR)-deficient human fibroblasts are extremely sensitive to UV-induced apoptosis and this sensitivity correlated with the induction of the p53 tumour suppressor. However, we have also found that p53 can be protective against UV-induced apoptosis. Thus, prior to this study, it was not clear whether the induction of p53 in TCR-deficient fibroblasts contributed to their death. To address this issue, we have expressed human papillomavirus E6 (HPV-E6) in primary fibroblasts derived from patients affected with xeroderma pigmentosum (complementation groups A, B and C) and Cockayne syndrome (complementation group B). We found that TCR-deficient (XP-A, XP-B and CS-B) fibroblasts were more sensitive than TCR-proficient cells (XP-C and normal) to both UV light and cisplatin treatment and this increase in sensitivity was not p53 dependent. Importantly, HPV-E6 expression increased the sensitivity of TCR-proficient normal and XP-C fibroblasts to UV- and cisplatin-induced apoptosis. This increase in sensitivity correlated with a decrease in the capacity of HPV-E6 expressing cells to recover mRNA synthesis following UV-irradiation. Therefore, we propose that p53 protects against UV- and cisplatin-induced apoptosis in a TCR-dependent manner and that p53 does not contribute strongly to the induction of apoptosis in TCR-deficient fibroblasts.

Apoptosis↗
PubMed2001

Induction of ser15 and lys382 modifications of p53 by blockage of transcription elongation.

Blockage of transcription has been shown to induce the tumor suppressor p53 in human cells. We here show that RNA synthesis inhibitors blocking the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II, such as DRB and H7, induced rapid nuclear accumulation of p53 proteins that were not phosphorylated at ser15 or acetylated at lys382. In contrast, agents that inhibit the elongation phase of transcription, such as UV light, camptothecin or actinomycin D, induced the accumulation of nuclear p53 proteins that were modified at both of these sites. Furthermore, using a panel of DNA repair-deficient cells we show that persistent DNA lesions in the transcribed strand of active genes are responsible for the induction of the ser15 and lys382 modifications following UV-irradiation. We conclude that inhibition of transcription is sufficient for the accumulation of p53 in the nucleus regardless of whether the ser15 site of p53 is phosphorylated or not. Importantly, blockage of the elongation phase of transcription triggers a distinct signaling pathway leading to p53 modifications on ser15 and lys382. We propose that the elongating RNA polymerase complex may act as a sensor of DNA damage and as an integrator of cellular stress signals.

Acetylation↗
PubMed2001

UV light-induced degradation of RNA polymerase II is dependent on the Cockayne's syndrome A and B proteins but not p53 or MLH1.

It has been hypothesized that the degradation of the largest subunit of RNA polymerase II (polIILS) is required for transcription-coupled repair (TCR) of UV light-induced transcription-blocking lesions. In this study we further investigated the mechanism of UV-induced degradation of polIILS using cell lines with specific defects in TCR or in the recovery of RNA synthesis. It was found that the hypophosphorylated IIa form of polIILS rapidly decreased following UV-irradiation in all cell lines tested. Inhibition of proteasome activity resulted in an increase of the hyperphosphorylated IIo form of polIILS in UV-irradiated cells, while inhibition of CTD-kinases resulted in the retention of the IIa form. In UV-irradiated Cockayne's syndrome cells, which are defective in TCR, the levels of the IIo form increased in a similar manner as when proteasome inhibitors were added to UV-irradiated normal cells. In contrast, TCR-deficient HCT116 cells, which lack the mismatch repair protein MLH1, showed proficient degradation of polIILS as did cells with deficiencies in the recovery of RNA synthesis following UV-irradiation due to defective p53. Furthermore, we found that proteasome function was important for the recovery of mRNA synthesis even in TCR-deficient HCT116 cells. Our results suggest that proteasome-mediated degradation of polIILS is preceded by phosphorylation of the C-terminal domain of polIILS and requires the CS-A and CS-B but not MLH1 or p53 proteins. Furthermore, our results suggest that following UV-irradiation, the degradation of polIILS is required for the efficient recovery of mRNA synthesis but not for TCR per se.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress.

The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21(Waf1) gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes.

Blotting, Northern↗
PubMed2001

The cyclin-dependent kinase inhibitor roscovitine inhibits RNA synthesis and triggers nuclear accumulation of p53 that is unmodified at Ser15 and Lys382.

Roscovitine has been shown to induce the accumulation of the tumor suppressor p53, to arrest cells in the G(1) and G(2)/M phases of the cell cycle, and to induce apoptosis in human cells. Although these cellular effects of roscovitine are thought to be caused directly by its specific inhibition of cyclin-dependent kinases, other mechanisms may contribute as well. In this study, we investigated whether roscovitine interferes with transcription in human cells. We have previously shown that blockage of transcription is a trigger for the induction of p53 and apoptosis in human fibroblasts. Here we show that mRNA synthesis is suppressed significantly by roscovitine in human cells. Furthermore, our results suggest that the mechanism by which roscovitine inhibits RNA synthesis involves the inhibition of the phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Cells treated with roscovitine at doses that affected transcription were found to accumulate p53 in the nucleus; curiously, however, the nuclear accumulation of p53 was not accompanied by modifications at either the Ser15 or Lys382 sites of p53. We conclude that roscovitine is a potent inhibitor of RNA synthesis and that this inhibition may be responsible for the accumulation of nuclear p53.

CDC2-CDC28 Kinases↗
PubMed2000

The human Cdc14 phosphatases interact with and dephosphorylate the tumor suppressor protein p53.

The yeast Cdc14 phosphatase has been shown to play an important role in cell cycle regulation by dephosphorylating proteins phosphorylated by the cyclin-dependent kinase Cdc28/clb. We recently cloned two human orthologs of the yeast CDC14, termed hCDC14A and -B, the gene products of which share approximately 80% amino acid sequence identity within their N termini and phosphatase domains. Here we report that the hCdc14A and hCdc14B proteins interact with the tumor suppressor protein p53 both in vitro and in vivo. This interaction is dependent on the N termini of the hCdc14 proteins and the C terminus of p53. Furthermore, the hCdc14 phosphatases were found to dephosphorylate p53 specifically at the p34(Cdc2)/clb phosphorylation site (p53-phosphor-Ser(315)). Our findings that hCdc14 is a cyclin-dependent kinase substrate phosphatase suggest that it may play a role in cell cycle control in human cells. Furthermore, the identification of p53 as a substrate for hCdc14 indicates that hCdc14 may regulate the function of p53.

Cell Cycle Proteins↗
PubMed2000

The tumor suppressor p53 can both stimulate and inhibit ultraviolet light-induced apoptosis.

We have previously shown that the tumor suppressor p53 can play a protective role against UV-induced apoptosis in human fibroblasts. In the present study, we investigated whether the protective function of p53 expression is established before or after UV irradiation. Using a stable human cell line expressing a murine temperature-sensitive p53 in which p53 function could be tightly and reversibly regulated, we found that functional p53 stimulated the induction of apoptosis when expressed for as little as 4-12 h after UV irradiation and that this induction was not dependent on de novo protein synthesis. In contrast, expression of p53 for 12 h or more before UV irradiation reduced the extent of apoptosis even when functional p53 expression was maintained after irradiation. The protection conferred by p53 required ongoing protein synthesis and correlated with enhanced recovery of mRNA synthesis. Together, these results suggest that p53 induces distinct proapoptotic and antiapoptotic signals and that these opposing activities can be separated both temporally and by their requirement for de novo protein synthesis. These findings may have important implications for the refinement of gene therapy approaches combining p53 with pharmacological agents that target transcription or translation.

Apoptosis↗
PubMed2000

Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors.

In this study, we explored what effect inhibitors of the 26S proteasome have on cell cycle distribution and induction of apoptosis in human skin fibroblasts and colon cancer cells differing in their p53 status. We found that proteasome inhibition resulted in nuclear accumulation of p53. This was surprising because it is thought that the degradation of p53 is mediated by cytoplasmic 26S proteasomes. Nuclear accumulation of p53 was accompanied by the induction of both p21WAF1 mRNA and protein as well as a decrease in cells entering S phase. Interestingly, cells with compromised p53 function showed a marked increase in the proportion of cells in the G2-M phase of the cell cycle and an attenuated induction of apoptosis after proteasome inhibition. Taken together, our results suggest that proteasome inhibition results in nuclear accumulation of p53 and a p53-stimulated induction of both G1 arrest and apoptosis.

Acetylcysteine↗
PubMed1999

Inhibition of RNA polymerase II as a trigger for the p53 response.

The mechanisms by which the p53 response is triggered following exposure to DNA-damaging agents have not yet been clearly elucidated. We and others have previously suggested that blockage of RNA polymerase II may be the trigger for induction of the p53 response following exposure to ultraviolet light. Here we report on the correlation between inhibition of mRNA synthesis and the induction of p53, p21WAF1 and apoptosis in diploid human fibroblasts treated with either UV light, cisplatin or the RNA synthesis inhibitors actinomycin D, DRB, H7 and alpha-amanitin. Exposure to ionizing radiation or the proteasome inhibitor LLnL, however, induced p53 and p21WAF1 without affecting mRNA synthesis. Importantly, induction of p53 by the RNA synthesis or proteasome inhibitors did not correlate with the induction of DNA strand breaks. Furthermore, cisplatin-induced accumulation of active p53 in repair-deficient XP-A cells occurred despite the lack of DNA strand break induction. Our results suggest that the induction of the p53 response by certain toxic agents is not triggered by DNA strand breaks but rather, may be linked to inhibition of mRNA synthesis either directly by the poisoning of RNA polymerase II or indirectly by the induction of elongation-blocking DNA lesions.

Apoptosis↗
PubMed1999

Role for p53 in the recovery of transcription and protection against apoptosis induced by ultraviolet light.

We have previously suggested that the inhibition of RNA polymerase II-mediated transcription after exposure to UV light promotes the accumulation of p53 and the induction of apoptosis (Oncogene 13, 823-831). However, it was not clear whether p53 induction was contributing to apoptosis. Here we report that apoptosis is triggered at lower UV doses in p53-deficient Li-Fraumeni syndrome (LFS) and human papillomavirus (HPV) E6 expressing fibroblasts than in normal cells, suggesting that p53 can be protective against UV-induced apoptosis. There is no significant difference in the effect of UV-irradiation on the cell cycle distribution of normal and primary LFS fibroblasts. Importantly, the recovery of nascent mRNA synthesis in all p53-deficient fibroblasts is significantly impaired compared with control cells after exposure to relevant doses of UV light. Taken together, our results suggest that wild-type p53 can protect cells against UV-induced apoptosis by facilitating the recovery of transcription. Furthermore, we suggest that the capacity of cells to recover transcription after genotoxic damage is an important determinant of sensitivity to apoptosis.

Apoptosis↗
PubMed1999

Recovery of RNA synthesis from the DHFR gene following UV-irradiation precedes the removal of photolesions from the transcribed strand.

It is thought that recovery of RNA synthesis following UV-irradiation is closely related to the removal of UV-induced lesions from the transcribed strand of active genes. To test this hypothesis, nascent RNA synthesis from three different locations within the DHFR gene in CHO cells was assessed following exposure to UV light (254 nm). Using both in vivo RNA labeling as well as the nuclear run-on technique, it was found that RNA synthesis from the middle and the 3'-end of the gene was inhibited within 20 min by approximately 30 and 70%, respectively, while RNA synthesis from the 5'-end of the DHFR gene was enhanced. RNA synthesis from the middle portion of the gene fully recovered within 30-45 min of post-UV incubation, while recovery was slower from the 3'-end of the gene. Compared with previously published data for the kinetics of removal of UV-induced DNA lesions from the 5'-half of the DHFR gene in these cells, it is concluded that RNA synthesis resumed significantly faster in this region than could be accounted for by the removal of photolesions from the transcribed strand. Thus, although RNA synthesis was initially inhibited by UV-induced photolesions, the results suggest that RNA polymerase II was able to bypass these lesions prior to their removal.

Animals↗
PubMed1999

Potential roles for p53 in nucleotide excision repair.

Ultraviolet (UV) light-induced DNA damage is repaired by the nucleotide excision repair pathway, which can be subdivided into transcription-coupled repair (TCR) and global genome repair (GGR). Treatment of cells with a priming dose of UV light appears to stimulate both GGR and TCR, suggesting that these processes are inducible. GGR appears to be disrupted in p53-deficient fibroblasts, whereas the effect of p53 disruption on TCR remains somewhat controversial. Normal recovery of mRNA synthesis following UV irradiation is thought to depend on TCR. We have found that the recovery of mRNA synthesis following exposure to UV light is severely attenuated in p53-deficient human fibroblasts. Therefore, p53 disruption may lead to a defect in TCR or a post-repair process required for the recovery of mRNA synthesis. Several different functions of p53 have been proposed which could contribute to these cellular processes. We suggest that p53 could participate in GGR and the recovery of mRNA synthesis following UV exposure through the regulation of steady-state levels of one or more p53-regulated gene products important for these processes. Furthermore, we suggest that the role of p53 in the recovery of mRNA synthesis is important for resistance to UV-induced apoptosis.

Cockayne Syndrome↗
PubMed1999

Dose-dependent effects of DNA-damaging agents on p53-mediated cell cycle arrest.

We examined the dose-dependent effects of DNA-damaging agents on G1 arrest in isogenic human cell lines differing in their p53 status. As expected, 5 or 20 Gy of ionizing radiation induced a p53-dependent G1 arrest. In contrast, UV light or actinomycin D induced a modest G1 arrest that was p53-dependent only at lower doses. At higher doses, cells were arrested in G1 in a p53-independent manner coinciding with inhibition of RNA synthesis and abolished cyclin E expression. Interestingly, expression of cyclin E was enhanced after exposure to moderate doses of UV light and actinomycin D, and this enhancement was suppressed by wild-type p53. We propose that agents inducing transcription-blocking DNA lesions will at higher doses inhibit the progression of cells into S phase by a p53-independent mechanism involving the attenuation of E2F-mediated transcription of genes, such as cyclin E.

Blotting, Western↗
PubMed1999

Repair of radiation-induced DNA strand breaks does not occur preferentially in transcriptionally active DNA.

Certain DNA base lesions induced by ionizing radiation or oxidative stress are repaired faster from the transcribed strand of active genes compared to the genome overall. In this study, it was investigated whether radiation-induced DNA strand breaks are preferentially repaired in active genes compared to the genome as a whole in CHO cells. The alkaline unwinding technique coupled to slot-blot hybridization with specific DNA probes was used to study the induction and repair of DNA strand breaks in defined DNA sequences. Results using this technique showed a linear dose response for the formation of radiation-induced DNA strand breaks in the dihydrofolate reductase (DHFR) gene. Furthermore, the half-life of radiation-induced strand breaks was less than 5 min in the DHFR gene, in the ribosomal genes, and in the genome as a whole. These results suggest that the repair of DNA strand breaks is fast and uniform in the genome of mammalian cells.

Animals↗
PubMed1998

Persistent DNA damage induced by ultraviolet light inhibits p21waf1 and bax expression: implications for DNA repair, UV sensitivity and the induction of apoptosis.

Ultraviolet light (UV) induced DNA lesions efficiently block transcript elongation and induce the p53 response. Although p53 contributes to transcriptional activation of the p21waf1 and bax genes, accumulation of these proteins requires that these genes are free of UV induced pyrimidine dimers. We assessed the level of expression of p53 and the p53 regulated p21waf1 and bax gene products in normal diploid fibroblasts (NDF) and several nucleotide excision repair deficient fibroblasts following UV-irradiation. At low UV fluences, increased expression of p53, p21waf1 and bax was only observed in fibroblasts deficient in transcription coupled repair (TCR). Whereas p53 protein levels increased in all cell types at high UV fluences, p21waf1 levels initially decreased and then recovered in a manner dependent on TCR. At later times, expression of p21waf1 and bax was only elevated in TCR-proficient cells. The lack of TCR strongly correlated with an enhanced induction of apoptosis. Furthermore, we assessed the effect of modulation of the p53/p21waf1/pRb pathway on clonogenic survival following UV irradiation. Expression of E2F-1, E2F-4, and the large tumour antigens of SV40 and Polyomavirus conferred UV sensitivity to NDF whereas p21waf1 protected cells against UV treatment. We propose that the fluence dependent attenuation of protective functions of p53 by blockage of transcription favours apoptosis following UV exposure.

Apoptosis↗
PubMed1998

Butyrate attenuates BCLX(L) expression in human fibroblasts and acts in synergy with ionizing radiation to induce apoptosis.

Ionizing radiation is a poor inducer of apoptosis in many cell types. In this study we investigated what effect the differentiation agent butyrate had on the cellular levels of the apoptosis regulators BCL2, BCLX(L) and BAX and on radiation-induced apoptosis. It was found that butyrate significantly lowered the level of the apoptosis antagonist BCLX(L) in a time- and dose-dependent manner in diploid human fibroblasts. The reduction of the level of BCLX(L) protein by butyrate correlated with an increased induction of apoptosis in cells irradiated with ionizing radiation. Butyrate also acted in synergy with ultraviolet (UV) light and cisplatin to induce apoptosis in human fibroblasts. Radiosensitization was obtained when butyrate was added before and/or after irradiation, although the combination of treatment both before and after irradiation was the most effective. Our results suggest that butyrate acts in synergy with ionizing radiation, UV light and cisplatin to induce apoptosis, perhaps by lowering the level of the apoptosis antagonist BCLX(L) in cells.

Apoptosis↗