Analysis of HPV-16 early gene regulationin cellular differentiation, includingcharacterisation of the possiblerole of CPEB proteins

Christina Neigaard Hansen

Analysis of HPV-16 early gene regulationin cellular differentiation, includingcharacterisation of the possiblerole of CPEB proteins

Christina Neigaard Hansen

Abstract

Livmoderhalskræft er den næst hyppigste diagnosticerede cancerform i kvinder på verdensplan. Canceren menes at være forårsaget af infektioner med høj-risiko humane papillomavira (HPVs), hvor HPV-16 er den type blandt de høj-risiko typer, der oftest identificeres i livmoderhalskræft. HPV-16 koder for 6 tidlige og 2 sene proteiner (E1, E2, E4, E5, E5, E6, E7, L1 og L2), hvoraf E6 og E7 er de vigtigste oncogener. I vores laboratorium er det tidligere blevet vist, at den mest distale del af E6/E7 messengerne fra flere HPV typer nedregulerer protein syntesen af et reporter gen via 4 cytoplasmatiske polyadenylerings elementer (CPEs) placeret i den distale del af messengerne. Disse CPE sekvenser binder det CPE-bindende protein CPEB. I dette studie er mRNA niveauerne af de 4 CPEB'er blevet analyseret i primære keratinocytter, i 8 forskellige celle linier samt både i normale og cancer genitale væv. Store variationer var observeret både blandt de forskellige celle typer og blandt de 4 CPEB'er. Et interessant fund i ovarie cancer var nedregulering i mRNA niveauet af CPEB1, et protein der tidligere er blevet foreslået til at være et tumor suppressor protein. Vi fandt også en tendens til nedregulering af CPEB3 mRNA i livmoderhalskræft, hvilket måske kunne påvirke reguleringen af E6/E7 messengerer under carcinogenesen. De to hyppige brugte interne reference markører GAPDH og U6snRNA var opreguleret i cancer væv. Derudover analyserede vi aktiviteten af promotorer, der regulere HPV-16 E6/E7 ekspressionen. HPV-16 inficerer fortrinsvis de proliferende celler i det konstant fornyende flerlagede epitel, der beklæder livmoderhalsen. Disse proliferende celler differentiere, mens de bliver skubbet opad i epitelet af nye datter celler. Virusens livs cyklus er tæt koblet til dette differentierings program, hvor de tidlige gener hovedsageligt udtrykkes i den nedre del af epitelet og sene gener i de mere differentierede celler. Vi inducerede keratinocytterne til at differentiere, for derefter at måle aktiviteten af de forskellige promotorer man maner initierer E6/E7 transcriptionen. De analyserede promotorer viste alle aftagende aktivitet under differentiering. P542 promotoren, som vi tidligere har identificeret i E6 læserammen opstrøms for E7 læserammen, var dog ikke lige så stærkt påvirket af differentieringen som promotorerne i den lange kontrol region (LCR) opstrøms for E6 læserammen. Desuden viste vi transcription fra regioner indeholdende de knap så velbeskrevne transcriptions start steder ved nt.7436 og nt.7841 i LCR.

Originalsprog	Engelsk

Forlag	Museum Tusculanum
Status	Udgivet - 2008

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title = "Analysis of HPV-16 early gene regulationin cellular differentiation, includingcharacterisation of the possiblerole of CPEB proteins",

abstract = "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.",

author = "Hansen, {Christina Neigaard}",

year = "2008",

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T1 - Analysis of HPV-16 early gene regulationin cellular differentiation, includingcharacterisation of the possiblerole of CPEB proteins

AU - Hansen, Christina Neigaard

PY - 2008

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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 -

Analysis of HPV-16 early gene regulationin cellular differentiation, includingcharacterisation of the possiblerole of CPEB proteins

Abstract

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