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Human Papillomavirus (HPV) E7 Induces Prolonged G2 following S Phase Reentry in Differentiated Human Keratinocytes

Banerjee, N. Sanjib, Wang, Hsu-Kun, Broker, Thomas R., Chow, Louise T.·2011·Journal of Biological Chemistry
DOI 10.1074/jbc.m110.197574
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FIGURE 1 HPV-18 E7 and genomic HPV-18 plasmid induce elevated expression of cyclin B1 and cdc2 in the differentiated strata of PHK raft cultures. A, double indirect immunofluorescence microscopy was performed to detect cyclin B1 ( green ) and BrdU ( red ) in organotypic raft cultures of PHK ( left panel ), HPV-18 E7 wild type ( middle panel ), and HPV-18 E7E35Q , E36Q , E37Q ( right panel ). B , cdc2 ( green ) and cyclin B1 ( red ) in raft cultures of HPV-18 E7 𝚫 DLLC ( upper row ), the wild type HPV-18 E7 ( middle row ), and HPV-18 genomic plasmid-containing raft cultures ( lower row ). 𝚫 DLLC and E35Q , E36Q , E37Q are mutations of HPV-18 E7 lacking the pocket protein binding site or CKII motif, respectively. In this figure and Figs. 3 and 5, images were captured at × 20 magnification and arrowheads indicate the basal layer of the epithelia. All the raft cultures shown here are 10 days old.
FIGURE 1 HPV-18 E7 and genomic HPV-18 plasmid induce elevated expression of cyclin B1 and cdc2 in the differentiated strata of PHK raft cultures. A, double indirect immunofluorescence microscopy was performed to detect cyclin B1 ( green ) and BrdU ( red ) in organotypic raft cultures of PHK ( left panel ), HPV-18 E7 wild type ( middle panel ), and HPV-18 E7E35Q , E36Q , E37Q ( right panel ). B , cdc2 ( green ) and cyclin B1 ( red ) in raft cultures of HPV-18 E7 𝚫 DLLC ( upper row ), the wild type HPV-18 E7 ( middle row ), and HPV-18 genomic plasmid-containing raft cultures ( lower row ). 𝚫 DLLC and E35Q , E36Q , E37Q are mutations of HPV-18 E7 lacking the pocket protein binding site or CKII motif, respectively. In this figure and Figs. 3 and 5, images were captured at × 20 magnification and arrowheads indicate the basal layer of the epithelia. All the raft cultures shown here are 10 days old.CC-BY · From the PDF
FIGURE 2 HPV-18 E7 andgenomicHPV-18plasmiddysregulate cell cycle regulatory proteins favoring a G2 phase milieu. A , FACS analysis of HeLa cells without any treatment or treated with hydroxyurea ( HU ) to enrich cells in S phase or with nocodazole ( Noc ), which enrich cells in M. B-E , Western blot analysis of lysates from asynchronous ( AS ) HeLa, hydroxyurea-treated HeLa, vector-transduced raft culture ( Vector ), HPV-18 E7 -expressing raft culture ( 18E7 ), PHK raft culture containing amplifying HPV-18 genomic plasmids ( HPV-18 ), PHK control raft culture ( PHK ), and Noc-treated HeLa. B , immunoblots showing steady state levels of HPV-18 E7, cyclin B1, p -cdc2 Thr 14 , and p -cdc2 Thr 161 . C , steady state expression levels of p -cdc2 Tyr 15 and p -cdc2 Thr 161 . D , steady state expression levels of cdc2 and phospho-histone Ser 10 (p-H3S10). E , steady state expression level of Wee1. F , steady state expression level of unphosphorylated Myt1 ( upper panel ) and phosphorylated Myt1 ( middle panel ). B-D and F represent 4 different gels. C and E are from the same gel. The blot represented in panel B wasprobed, stripped, and reprobed with different antibodies. Actin was used as a protein loading control.
FIGURE 2 HPV-18 E7 andgenomicHPV-18plasmiddysregulate cell cycle regulatory proteins favoring a G2 phase milieu. A , FACS analysis of HeLa cells without any treatment or treated with hydroxyurea ( HU ) to enrich cells in S phase or with nocodazole ( Noc ), which enrich cells in M. B-E , Western blot analysis of lysates from asynchronous ( AS ) HeLa, hydroxyurea-treated HeLa, vector-transduced raft culture ( Vector ), HPV-18 E7 -expressing raft culture ( 18E7 ), PHK raft culture containing amplifying HPV-18 genomic plasmids ( HPV-18 ), PHK control raft culture ( PHK ), and Noc-treated HeLa. B , immunoblots showing steady state levels of HPV-18 E7, cyclin B1, p -cdc2 Thr 14 , and p -cdc2 Thr 161 . C , steady state expression levels of p -cdc2 Tyr 15 and p -cdc2 Thr 161 . D , steady state expression levels of cdc2 and phospho-histone Ser 10 (p-H3S10). E , steady state expression level of Wee1. F , steady state expression level of unphosphorylated Myt1 ( upper panel ) and phosphorylated Myt1 ( middle panel ). B-D and F represent 4 different gels. C and E are from the same gel. The blot represented in panel B wasprobed, stripped, and reprobed with different antibodies. Actin was used as a protein loading control.CC-BY · From the PDF
FIGURE 3 HPV-18 E7 and genomic HPV-18 plasmid induce changes in the expression and phosphorylation status of the cdc25C phosphatase. A , indirect immunofluorescence microscopy was used to detect the expression and localization of p -cdc25C Ser 216 ( red ) relative to cdc2 ( green ) in raft cultures of HPV-18 E7 , HPV-18 E7 𝚫 DLLC , HPV-18 E7C27S , HPV-18 E7S32Q , S33Q , and HPV-18 genomic plasmids. B , immunoblot analysis were performed on lysates from vector-only PHK raft cultures, HPV-18 E7-expressing raft cultures, and HPV-18 raft cultures to assess total cdc25C and the phosphorylated cdc25C Ser 216 . C , simultaneous indirect immunofluorescence and HPV-18 DNA fluorescent in situ hybridization to detect HPV-18 DNA amplification ( red ) relative to p -cdc25C Ser 216 ( green ) or cdc2 ( green ). E7C27S is mutated in the pocket protein binding motif, whereas S32Q,S33Q is mutated in the CKII substrates. Raft cultures in A and B are 10 days old and raft culture in C is 12 days old.
FIGURE 3 HPV-18 E7 and genomic HPV-18 plasmid induce changes in the expression and phosphorylation status of the cdc25C phosphatase. A , indirect immunofluorescence microscopy was used to detect the expression and localization of p -cdc25C Ser 216 ( red ) relative to cdc2 ( green ) in raft cultures of HPV-18 E7 , HPV-18 E7 𝚫 DLLC , HPV-18 E7C27S , HPV-18 E7S32Q , S33Q , and HPV-18 genomic plasmids. B , immunoblot analysis were performed on lysates from vector-only PHK raft cultures, HPV-18 E7-expressing raft cultures, and HPV-18 raft cultures to assess total cdc25C and the phosphorylated cdc25C Ser 216 . C , simultaneous indirect immunofluorescence and HPV-18 DNA fluorescent in situ hybridization to detect HPV-18 DNA amplification ( red ) relative to p -cdc25C Ser 216 ( green ) or cdc2 ( green ). E7C27S is mutated in the pocket protein binding motif, whereas S32Q,S33Q is mutated in the CKII substrates. Raft cultures in A and B are 10 days old and raft culture in C is 12 days old.CC-BY · From the PDF
FIGURE 4 HPV-18 E7 and genomic HPV-18 plasmid activate kinases responsible for cdc25C phosphorylation on Ser 216 . Immunoblot analysis of ( A ) p -ATR Ser 428 , p -Chk1 Ser 345 , total Chk2, and p -Chk2 Thr 68 and ( B ) ATM, p -ATMSer 1981 , total Chk1 from lysates of raft cultures harboring empty vector ( Vector ), HPV-18 genomic plasmid, or expressing HPV-18 E7 ( 18E7 ) or HPV-18 E7 𝚫 DLLC ( 𝚫 DLLC ) mutation; C , JNK2 and phosphorylated JNKs in raft cultures harboring the vector-only ( Vector ), HPV-18 E7 ( 18E7 ), E7 𝚫 DLLC ( 𝚫 DLLC ), E7S32Q , E33Q ( S32Q,S33Q ), or HPV-18 genomic plasmids. HeLa ( Noc ) denotes lysates from nocodazole-treated HeLa as negative control for JNK2.
FIGURE 4 HPV-18 E7 and genomic HPV-18 plasmid activate kinases responsible for cdc25C phosphorylation on Ser 216 . Immunoblot analysis of ( A ) p -ATR Ser 428 , p -Chk1 Ser 345 , total Chk2, and p -Chk2 Thr 68 and ( B ) ATM, p -ATMSer 1981 , total Chk1 from lysates of raft cultures harboring empty vector ( Vector ), HPV-18 genomic plasmid, or expressing HPV-18 E7 ( 18E7 ) or HPV-18 E7 𝚫 DLLC ( 𝚫 DLLC ) mutation; C , JNK2 and phosphorylated JNKs in raft cultures harboring the vector-only ( Vector ), HPV-18 E7 ( 18E7 ), E7 𝚫 DLLC ( 𝚫 DLLC ), E7S32Q , E33Q ( S32Q,S33Q ), or HPV-18 genomic plasmids. HeLa ( Noc ) denotes lysates from nocodazole-treated HeLa as negative control for JNK2.CC-BY · From the PDF
FIGURE 5 A , indirect immunofluorescent detection of p -ATMSer 1981 ( green ) and S phase reentry (BrdU incorporation, red ) in vector-only transduced PHK raft culture ( top row ), HPV-18 E7 𝚫 DLLC mutation-expressing raft culture ( second row ), HPV-18 E7-expressing raft culture ( third row ), and HPV-18 plasmid containing raft culture ( fourth row ). In the last row, the red arrow indicates the high BrdU and low p -ATMSer 1981 , whereas the green arrow points to high p -ATMSer 1981 but low or no BrdU incorporation. B , p -ATMSer 1981 expression relative to HPV-18 plasmid amplification was revealed by combined indirect immunofluorescence andHPV-18DNAfluorescent in situ hybridization. Redarrow indicate high HPV-18 DNA and low or no p -ATMSer 1981 , whereas the greenarrow points to the high p -ATM Ser 1981 but low or no HPV-18 plasmid. Raft cultures in A are 10 days old and raft culture in B is 12 days old.
FIGURE 5 A , indirect immunofluorescent detection of p -ATMSer 1981 ( green ) and S phase reentry (BrdU incorporation, red ) in vector-only transduced PHK raft culture ( top row ), HPV-18 E7 𝚫 DLLC mutation-expressing raft culture ( second row ), HPV-18 E7-expressing raft culture ( third row ), and HPV-18 plasmid containing raft culture ( fourth row ). In the last row, the red arrow indicates the high BrdU and low p -ATMSer 1981 , whereas the green arrow points to high p -ATMSer 1981 but low or no BrdU incorporation. B , p -ATMSer 1981 expression relative to HPV-18 plasmid amplification was revealed by combined indirect immunofluorescence andHPV-18DNAfluorescent in situ hybridization. Redarrow indicate high HPV-18 DNA and low or no p -ATMSer 1981 , whereas the greenarrow points to the high p -ATM Ser 1981 but low or no HPV-18 plasmid. Raft cultures in A are 10 days old and raft culture in B is 12 days old.CC-BY · From the PDF

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Metadata

Authors
Banerjee, N. S., Wang, H.-K., Broker, T. R., & Chow, L. T.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2011-04
Volume
286
Issue
17
Pages
15473-15482
ISSN
0021-9258
DOI
10.1074/jbc.m110.197574
Cited by
97
References
2
Language
en
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CoChat. (2026). Citation check: Human Papillomavirus (HPV) E7 Induces Prolonged G2 following S Phase Reentry in Differentiated Human Keratinocytes. https://app.cochat.ai/papers/10.1074/jbc.m110.197574
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Banerjee, N. S., Wang, H.-K., Broker, T. R., & Chow, L. T. (2011). Human Papillomavirus (HPV) E7 Induces Prolonged G2 following S Phase Reentry in Differentiated Human Keratinocytes. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.m110.197574