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Suppressive effects of p53 protein on heat-induced centrosomal abnormality
International Congress Series 1236 (2002) 375 – 377
Suppressive effects of p53 protein on heat-induced centrosomal abnormality Mana Miyakoda, Keiji Suzuki, Seiji Kodama, Masami Watanabe * Laboratory of Radiation and Life Science, School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
Abstract Accumulation and activation of the p53 protein is necessary for maintenance of genome stability in response to DNA strand breaks. Recently, it has been found that heat shock, which does not directly damage DNA, induces accumulation of p53. However, it is not clear whether activated p53 suppresses heat-induced genome destabilization. Here, we examined centrosome destabilization by heat. We found that centrosomal abnormality, including denatured, dispersed and multiple centrosomes, increased after heat shock at 43 jC for 2 h. Although the frequency of centrosomal abnormality was not significantly deferent in p53 functional and dysfunctional cells, the cells with multiple centrosomes were found to grow in p53-dysfunctional cells. These results suggest that p53 has no effect on the induction of heat-induced centrosome damage; however, it suppresses the growth of cells with abnormal centrosomes. This study also showed that heat-induced centrosome dysfunction leads to abnormal separation of the chromosomes, and it may destabilize the chromosome number. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Heat shock; p53; Centrosome; Chromosome; Genome destabilization
1. Background DNA-damaging agents, such as ionising radiation, accumulate and activate the p53 protein as a transcriptional factor. Activated p53 induces cell cycle arrest or apoptosis, leading to inhibition of genome destabilization [1]. Recently, we have found that heat shock, which does not damage DNA, also accumulates and activates p53 [2]. However,
*
Corresponding author. Tel./fax: +81-95-844-5504. E-mail address: [email protected] (M. Watanabe).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 3 1 0 - 2
376
M. Miyakoda et al. / International Congress Series 1236 (2002) 375–377
the biological significance of p53 activation after heat shock is not clear. In the present study, we examined the protective effects of p53 on centrosomes, which maintain stability of the genome, after heat shock.
2. Materials and methods Normal human diploid fibroblasts (HE49), p53-functional tumour (HT1080 and MCF7) and p53 non-functional tumour (T24, RD, A431 and H1299) were heat shocked at 43 jC for 2 h. Immunofluorescence staining using the anti-gamma tubulin antibody was performed to visualize the centrosomes. The nucleus and chromosomes were stained with Giemsa’s solution.
3. Results To determine whether p53 plays a role in heat-shocked cells, we first examined the nuclear morphology of the p53-null NCI-H1299 cells. We found that the cells had multi or giant nuclei, resulting from abnormal chromosome separation. Therefore, we next examined centrosome abnormality, which may cause abnormal chromosome separation after heat shock. While one or two centrosomes are detected in cells at the G1 phase or S and G2 phase in non-treated cells, respectively, three types of centrosome abnormality were observed by immunostaining. One was the decreased staining of centrosomes, one was dispersed centrosomes and the last one was the multiple centrosomes. We compared the frequency of these centrosome abnormalities in p53-functional and dysfunctional cells. Twelve hours after heat shock was carried out, more than 40% of the cells showed decreased staining intensity of centrosomes, in all types of cells. The most probable cause is centrosome denaturation or degradation. In fact, the centrosomes became visible but the percentage of cells containing multiple centrosomes was increased thereafter. In addition, we could not find any significant differences between the p53 function-deficient cells (T24, A431, RD, and H1299) and cells with functional p53 (HE49, MCF7, HT1080). We also counted the percentage of colonies in which more than 50% of the cells contained multiple centrosomes, 120 h after heat shock. The percentage was more than 40% in the p53dysfunctional cells; however, it was less in the p53 functional cells. These results indicate that cells with multiple centrosomes may proliferate in the absence of functional p53.
4. Conclusion The present study indicates that heat shock denatures or degrades centrosomes immediately after treatment. Although the damaged centrosomes become visible, the number is increased. The proliferation of cells with multiple centrosomes is suppressed by functional p53; otherwise, cells with an abnormal number of chromosomes should increase. It is suggested that activation of p53 by heat shock may result in inhibition of genome destabilization.
M. Miyakoda et al. / International Congress Series 1236 (2002) 375–377
377
Acknowledgements This work was supported by a grant of Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References [1] D.B. Woods, K.H. Vousden, Regulation of p53 function, Exp. Cell Res. 264 (1) (2001) 56 – 66. [2] M. Miyakoda, K. Suzuki, S. Kodama, M. Watanabe, Heat induced G1 arrest is dependent on p53 function but not RB dephosphorylation, Biochem. Biophys. Res. Commun. 266 (1999) 377 – 381.
Suppressive effects of p53 protein on heat-induced centrosomal abnormality Mana Miyakoda, Keiji Suzuki, Seiji Kodama, Masami Watanabe * Laboratory of Radiation and Life Science, School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
Abstract Accumulation and activation of the p53 protein is necessary for maintenance of genome stability in response to DNA strand breaks. Recently, it has been found that heat shock, which does not directly damage DNA, induces accumulation of p53. However, it is not clear whether activated p53 suppresses heat-induced genome destabilization. Here, we examined centrosome destabilization by heat. We found that centrosomal abnormality, including denatured, dispersed and multiple centrosomes, increased after heat shock at 43 jC for 2 h. Although the frequency of centrosomal abnormality was not significantly deferent in p53 functional and dysfunctional cells, the cells with multiple centrosomes were found to grow in p53-dysfunctional cells. These results suggest that p53 has no effect on the induction of heat-induced centrosome damage; however, it suppresses the growth of cells with abnormal centrosomes. This study also showed that heat-induced centrosome dysfunction leads to abnormal separation of the chromosomes, and it may destabilize the chromosome number. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Heat shock; p53; Centrosome; Chromosome; Genome destabilization
1. Background DNA-damaging agents, such as ionising radiation, accumulate and activate the p53 protein as a transcriptional factor. Activated p53 induces cell cycle arrest or apoptosis, leading to inhibition of genome destabilization [1]. Recently, we have found that heat shock, which does not damage DNA, also accumulates and activates p53 [2]. However,
*
Corresponding author. Tel./fax: +81-95-844-5504. E-mail address: [email protected] (M. Watanabe).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 3 1 0 - 2
376
M. Miyakoda et al. / International Congress Series 1236 (2002) 375–377
the biological significance of p53 activation after heat shock is not clear. In the present study, we examined the protective effects of p53 on centrosomes, which maintain stability of the genome, after heat shock.
2. Materials and methods Normal human diploid fibroblasts (HE49), p53-functional tumour (HT1080 and MCF7) and p53 non-functional tumour (T24, RD, A431 and H1299) were heat shocked at 43 jC for 2 h. Immunofluorescence staining using the anti-gamma tubulin antibody was performed to visualize the centrosomes. The nucleus and chromosomes were stained with Giemsa’s solution.
3. Results To determine whether p53 plays a role in heat-shocked cells, we first examined the nuclear morphology of the p53-null NCI-H1299 cells. We found that the cells had multi or giant nuclei, resulting from abnormal chromosome separation. Therefore, we next examined centrosome abnormality, which may cause abnormal chromosome separation after heat shock. While one or two centrosomes are detected in cells at the G1 phase or S and G2 phase in non-treated cells, respectively, three types of centrosome abnormality were observed by immunostaining. One was the decreased staining of centrosomes, one was dispersed centrosomes and the last one was the multiple centrosomes. We compared the frequency of these centrosome abnormalities in p53-functional and dysfunctional cells. Twelve hours after heat shock was carried out, more than 40% of the cells showed decreased staining intensity of centrosomes, in all types of cells. The most probable cause is centrosome denaturation or degradation. In fact, the centrosomes became visible but the percentage of cells containing multiple centrosomes was increased thereafter. In addition, we could not find any significant differences between the p53 function-deficient cells (T24, A431, RD, and H1299) and cells with functional p53 (HE49, MCF7, HT1080). We also counted the percentage of colonies in which more than 50% of the cells contained multiple centrosomes, 120 h after heat shock. The percentage was more than 40% in the p53dysfunctional cells; however, it was less in the p53 functional cells. These results indicate that cells with multiple centrosomes may proliferate in the absence of functional p53.
4. Conclusion The present study indicates that heat shock denatures or degrades centrosomes immediately after treatment. Although the damaged centrosomes become visible, the number is increased. The proliferation of cells with multiple centrosomes is suppressed by functional p53; otherwise, cells with an abnormal number of chromosomes should increase. It is suggested that activation of p53 by heat shock may result in inhibition of genome destabilization.
M. Miyakoda et al. / International Congress Series 1236 (2002) 375–377
377
Acknowledgements This work was supported by a grant of Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References [1] D.B. Woods, K.H. Vousden, Regulation of p53 function, Exp. Cell Res. 264 (1) (2001) 56 – 66. [2] M. Miyakoda, K. Suzuki, S. Kodama, M. Watanabe, Heat induced G1 arrest is dependent on p53 function but not RB dephosphorylation, Biochem. Biophys. Res. Commun. 266 (1999) 377 – 381.