TY - BOOK
T1 - Analysis of HPV-16 early gene regulationin cellular differentiation, includingcharacterisation of the possiblerole of CPEB proteins
AU - Hansen, Christina Neigaard
PY - 2008
Y1 - 2008
N2 - Cervical cancer is the second most common cancer form diagnosed in women worldwide. The cancer is thought to be caused by infections with high-risk human papillomaviruses (HPVs), of which HPV-16 is the one of the high-risk types that most frequently are identified in cervical cancers. It encodes 6 early and 2 late proteins (E1, E2, E4, E5, E5, E6, E7, L1, and L2) of which E6 and E7 are the major oncogenes. In our laboratory it was previously found that the most distal part of the E6/E7 messengers from several HPV types downregulate protein synthesis of a reporter gene through the influence of 4 cytoplasmic polyadenylation elements (CPEs) situated in the distal part of the messengers. These CPE sequences bind the CPE-binding protein CPEB. In this study, the mRNA levels of the 4 CPEBs in primary keratinocytes, in 8 different cell lines, and in both normal and cancer genital tissues have been analysed. Huge variations among both the different cell types and the 4 CPEBs were observed. Interestingly, in ovarian cancer we found downregulated mRNA levels of CPEB1, a protein that previously has been suggested to be a tumor suppressor protein. We also found a tendency for the CPEB3 mRNA to be downregulated in cervical cancer, which perhaps could influence the regulation of E6/E7 messengers during carcinogenesis. In addition, the two widely used internal reference markers GAPDH and U6snRNA were shown to be upregulated in the cancer tissues. Furthermore, we analysed the activity of promoters regulating the HPV-16 E6/E7 expression. HPV-16 preferably infects the proliferating cells of the continually renewing stratified epithelium lining the genital tract. These proliferating cells will differentiate as they are pushed upwards in the epithelium by newly produced daughter cells. The virus life cycle is tightly coupled to this differentiation program with the early genes mainly being expressed in the lower part of the epithelium and later genes in the more differentiated cells. We induced keratinocytes to differentiate and then measured the activity of the different promoters thought to initiate E6/E7 transcription. The promoters analysed all showed decreased activity during differentiation. However, the P542 promoter, which we previously identified in the E6 ORF upstream of the E7 ORF, was not as strongly influenced by the differentiation as the promoters in the long control region (LCR) upfront of the E6 ORF. In addition, we showed transcription from regions containing the not well-described transcriptions start sites at nt.7436 and nt.7841 in the LCR.
AB - Cervical cancer is the second most common cancer form diagnosed in women worldwide. The cancer is thought to be caused by infections with high-risk human papillomaviruses (HPVs), of which HPV-16 is the one of the high-risk types that most frequently are identified in cervical cancers. It encodes 6 early and 2 late proteins (E1, E2, E4, E5, E5, E6, E7, L1, and L2) of which E6 and E7 are the major oncogenes. In our laboratory it was previously found that the most distal part of the E6/E7 messengers from several HPV types downregulate protein synthesis of a reporter gene through the influence of 4 cytoplasmic polyadenylation elements (CPEs) situated in the distal part of the messengers. These CPE sequences bind the CPE-binding protein CPEB. In this study, the mRNA levels of the 4 CPEBs in primary keratinocytes, in 8 different cell lines, and in both normal and cancer genital tissues have been analysed. Huge variations among both the different cell types and the 4 CPEBs were observed. Interestingly, in ovarian cancer we found downregulated mRNA levels of CPEB1, a protein that previously has been suggested to be a tumor suppressor protein. We also found a tendency for the CPEB3 mRNA to be downregulated in cervical cancer, which perhaps could influence the regulation of E6/E7 messengers during carcinogenesis. In addition, the two widely used internal reference markers GAPDH and U6snRNA were shown to be upregulated in the cancer tissues. Furthermore, we analysed the activity of promoters regulating the HPV-16 E6/E7 expression. HPV-16 preferably infects the proliferating cells of the continually renewing stratified epithelium lining the genital tract. These proliferating cells will differentiate as they are pushed upwards in the epithelium by newly produced daughter cells. The virus life cycle is tightly coupled to this differentiation program with the early genes mainly being expressed in the lower part of the epithelium and later genes in the more differentiated cells. We induced keratinocytes to differentiate and then measured the activity of the different promoters thought to initiate E6/E7 transcription. The promoters analysed all showed decreased activity during differentiation. However, the P542 promoter, which we previously identified in the E6 ORF upstream of the E7 ORF, was not as strongly influenced by the differentiation as the promoters in the long control region (LCR) upfront of the E6 ORF. In addition, we showed transcription from regions containing the not well-described transcriptions start sites at nt.7436 and nt.7841 in the LCR.
M3 - Ph.D. thesis
BT - Analysis of HPV-16 early gene regulationin cellular differentiation, includingcharacterisation of the possiblerole of CPEB proteins
PB - Museum Tusculanum
ER -
