Carcinogenesis vol.12 no.9 pp.1627 —1631, 1991
Immortalization of normal human oral keratinocytes with type 16 human papillomavirus
No-Hee Park1*24, Byung-Moo Min\ Sheng-lin Li1, Min Zhong Huang1, Henry M.Cherick1 and Jay Doniger3 'UCLA School of Dentistry and 2UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA 90024, USA and 3Georgetown University School of Medicine, Washington, DC 20007, USA ^ o whom correspondence should be sent at the Section of Oral Biology, UCLA School of Dentistry, 43-033 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90024, USA
Introduction Human papillomavirus (HPV*) infects oral mucosa and is associated with the development of benign epithelial tumors and hyperplasia of the oral cavity, such as squamous cell papillomas, focal epithelial hyperplasia, chondyloma acuminatum, verruca vulgaris and epithelial dysplasia (1—8). HPV is also linked to certain human malignancies. This association is based on the finding that up to 90% of the cancer tissues from genital lesions contained the viral DNA (9). Of the >60 genomes of HPV, types 16 (HPV-16) and 18 (HPV-18), as well as recently isolated types 31 (HPV-31) and 33 (HPV-33), are most frequently associated with cervical cancer (10-12). They are found in up to 90% of stage 3 of cervical intra-epithelial neoplasia as well as in two-thirds of all invasive cervical cancers. A number of cell lines derived from cervix have been shown to contain both HPV DNA (13,14) and RNA (13). Furthermore, continued expression of virus-encoded proteins corresponding to the E6/E7 open reading frames (ORFs) has also been demonstrated in some of these cell lines (15,16). In a high percentage of cervical carcinomas and in cell lines derived from these cancers, HPV-16 and HPV-18 DNAs have been found to
Primary culture of normal human oral keratinocytes (NHOKs) Excised retromolar tissue from the oral cavity of a healthy male volunteer (with human subject committee approval) was washed in calcium- and magnesium-free Hanks' balanced salt solution (CMF-HBSS, GIBCO, Grand Island, NY). The tissue was incubated in CMF-HBSS containing collagenase (type II; 1.0 mg/ml, Millipore Corp., New Bedford, MA) and dispase (grade II; 2,4 mg/ml, Boenringer-Mannheim, Indianapolis, IN) to separate the epithelium from the underlying mesenchyme for 60 min at 37 °C in an atmosphere of 95% air and 5% CO}. Separated epithelial sheets were then dissociated into single cells by incubation in trypsin with agitation at 37°C for 30 min. The cells were washed with PBS, resuspended with keratinocyte growth medium (KGM; supplemented with pituitary extract, Clonetics Corp., San Diego, CA), and plated on plastic at a density of 5 x 104 cells per 28 cm2 (60 mm Petri dish).
•Abbreviations: HPV, human papillomavirus (HPV-16, type 16 HPV, etc.); ORF, open reading frame; NHOK, normal human oral keratinocyte; HOK-16A and HOK-16B, human and keratinocytes 16A and 16B; CMF-HBSS, calciumand magnesium-free Hanks' balances salt solution; KGM, keratinocyte growth medium.
Recombinant plasmid pMHPV-16d, a head-to-tail dimer of HPV-16 DNA inserted into the BamHl cloning site of the plasmid pdMMT,,^, containing G418 expression marker, was constructed as described earlier (28). The plasmid was then transfected into Escheridiia coli 294 (Genentech, South San Franciso, CA), amplified and purified with conventional methodology.
© Oxford University Press
Materials and methods
1627
Downloaded from http://carcin.oxfordjournals.org/ at Emory University on June 9, 2014
Primary human oral keratinocytes were transformed by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA, and two transformed cell lines named human oral keratinocytes-16A and -16B (HOK-16A and HOK-166) were established. While normal cells and cells transfected with vector only exhibited a limited lifespan, the HOK-16A and HOK-16B lines demonstrated immortality and altered morphology from their normal counterpart. The HOK-16A and HOK-16B lines contained ~ 40 and ~ 25 copies of intact HPV-16 DNA as integrated form per cell respectively, and both cell lines expressed several viral specific poly(A+) RNAs. Notably these cell lines also overexpressed cellular myc proto-oncogene in comparison with the normal counterpart. However, the immortalized cell lines were not able to produce tumors in nude mice, indicating that the cells are partially transformed. The HOK-16A and HOK-16B lines are, therefore, useful for investigating the multistep molecular events of oral carcinogenesis.
be integrated into cellular chromosomes, while the viral DNAs are generally retained as extra chromosomal episomes in premalignant dysplastic lesions (17-19). As in cervical cancers, HPV is also positively correlated with human oral malignancies. This relationship is explained by up to 28% of the cancer tissues from oral biopsies containing die viral DNA (20). Since the epithelium of oral mucosa and that of the female genital tract are histologically similar and they are continuously challenged by many environmental factors, close association of HPV widi the development of oral malignancies is not surprising. The availability of recombinant HPV DNA has allowed the investigation of the transforming activity of HPV in cell culture systems, which has provided supportive evidence of the transforming capacity of HPV. Morphologic transformation of C127 cells with HPV types 1 (HPV-1) and 5 (HPV-5) has been demonstrated (21). Neoplastic transformation of NTH/3T3 cells has been shown widi recombination HPV-16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full length of the HPV-16 genome (22). Similarly, transformation of human skin fibroblasts and keratinocytes was established with cloned HPV-16 and HPV-18 DNA (23-26). More recently, transformation and immortalization of normal human exocervical and cervical epithelial cells—in vivo target cells for HPV infection in genital region—was demonstrated with recombinant HPV-16 and HPV-18 DNA (26,27). Although oral keratinocytes are one of the major target cells for HPV infection and HPV-induced tumorigenesis in humans, the in vitro transforming activity of HPV in human oral keratinocytes has never been studied because of the unavailability of a culture system. In the present study, we cultured monolayers of human oral keratinocytes, transformed the cells with the introduction of the recombinant HPV-16 DNA into die cells by using liposome, and characterized the biological properties of the cells. The morphology, immortality, copy number of HPV-16 DNA per transformed cell, viral gene expression and cellular myc gene expression of die transformed cells were investigated.
N.-H.Park et al. Transfection of NHOKs Approximately 70% confluent primary NHOKs grown in 60 mm Petri dishes were transfected with pMHPV-16d or pdMMT^ by using the Ljpofectin reagent (BRL life Technologies Inc., Gaithersberg, MD). The Lipofectin reagent is a 1:1 (w/w) liposome formulation of the cationk lipid jV-[l-(2,3-dioleyloxy)propyl]-A'M/V-trirnethylaminocriloride (DOTMA) and dileoyl phsophatidylethanolamine (DOPE) in water. When mixed with DNA, the reagent interacts with DNA to form a lipid-DNA complex with complete entrapment of DNA (29). For each 60 mm dish, a mixture of 10 ^g/50 pi of pMHPV-16d (or pdMMT,,^) and 35 (ig/50 fii of Lipofectin reagent was added to culture medium, dropwise, as uniformly as possible with gentle swirling. The cells were incubated for 24 h at 37°C. The medium was then replaced with fresh KGM, and the cultures were incubated for an additional 48 h. To select cells transfected with the pMHPV-16d or pdMMTIKO plasmid, the cells were incubated in culture medium containing 70 jig/ml of G418 (GIBCO, Grand Island, NY) for 2 days, and cultured in fresh KGM for 7 additional days before subculture. Two G418-resistant cell colonies transfected with pMHPV-16d were isolated, subcultured and named HOK-16A and HOK-16B lines. Three G418-resistant cell colonies transfected with were also separated.
Fig. 1. Microscopic features of Giemsa-stained monolayers of NHOKs (A) and immortal HOK-16A (B) and HOK-16B lines ( Q . The HOK-16A and HOK-16B lines show denser growth pattern and morphological alterations. X130.
Probes and radiolabeling Linear HPV-16 DNA insert (79 kb in size) separated from the pMHPV-16d, v-myc oncogene (ATCC, Rockville, MD), and the human /3-actin gene (from Dr L.Kedes, Stanford University, Palo Alto, CA) were used as probes. They were labeled with 32 P by using the multiprime DNA labeling system (Amersham). The specific radioactivity of the labeled probes was always > 5 x 10s c.p.m./^g of DNA. In vivo tumorigenicity of H0K-16A and HOK-16B NHOK, HOK-16A and HOK-16B monolayer cultures were trypsinized, resuspended in PBS and injected s.c. into 15 athymic nude mice (nulnu; 1 x 107 cells/0.1 ml per animal; five animals per cell type) 1 day after mice had been X-irradiated (300 R). All mice were injected on the right flank and monitored twice weekly for the appearance of tumors over a period of > 3 months.
Results Proliferation pattern and morphology ofHOK-16A and H0K-16B The transforming activity of pMHPV-16d, a recombinant plasmid 1628
4.4 Fig. 2. Southern blot hybridization of high mol. wt cellular DNA (from NHOKs, H0K-16A and HOK-16B) to ["P1HPV-16 DNA after digestion with BamHI. The first three lanes represent a construction experiment in which an amount of pMHPV-16d DNA equal to 5, 25 or 125 copies per cell was rruxed with BamHI-digested NHOK DNA.
Downloaded from http://carcin.oxfordjournals.org/ at Emory University on June 9, 2014
DNA and RNA analysis High mol. wt cellular DNA from NHOK, HOK-16A and HOK-16B lines was extracted with phenol/cruoroform/isoamyl alcohol (25:24:1) and ethanol precipitated (30). To determine the presence of HPV-16 DNA and its copy number per cell if present, HPV-16 DNA [corresponding to 5, 25 and 125 copies of viral DNA per cell with BamHI-digested of NHOK DNA (10 /ig) as earner] and BamHI-digested cellular DNA (10 ^g) extracted from NHOK, HOK-16A and HOK-16B were electrophoresed in 1% agarose gel. The gel was treated with 0.25 M HCl to partially depurinate the DNA and transferred to a ntirocellulose filter (Amersham, Arlington Heights, IL) by Southern blotting (30). The nitrocellulose filter was hybridized to "P-labeled 7.9 kb total HPV-16 DNA under stringent condition (50% formamide in the presence of 10% dextran sulfate at 42°C for 16 h). The filter was washed twice in 2 x SSC buffer (1 x SSC: 150 mM NaCl plus 15 mM sodium citrate, pH 7.0) containing 0.5% SDS for 30 min at room temperature and twice in 0.1 x SSC containing 0.1 % SDS at 50°C for 30 min and exposed to Kodak SB-5 X-ray film (Eastman Kodak Co , Rochester, NY) at -70°C for 24 h. The density of the hybridized HPV-16 DNA bands was analyzed with a computing densitometer (Molecular Dynamics, Sunnyvale, CA). To determine the physical state of viral DNA in the immortalized HOK-16A and HOK-16B lines, 10 jig of high md. wt cellular DNA was digested with BamHl (B) and/or EcoRV (E) restriction enzymes. BomHI enzyme separates vector from HPV-16 sequences, while EcoRV does not digest pMHPV-16d. The fragmented DNAs were then transferred to nitrocellulose filter and hybridized to KP-labeled 7.9 kd HPV-16 DNA under stringent conditions. After washing the filter was exposed to X-ray film. To determine the expression of HPV DNA and c-myc proto-oncogene from NHOK and the immortalized cell lines, cellular poly(A + ) RNA was extracted from the cells and analyzed with Northern blot hybridization (30). Confluent NHOK, HOK-16A and HOK-16B lines were lysed with cold guanidine isothiocyanate. The lysatcs were layered onto CsCl gradient and centrifuged at 53 000 r.p.m. for 24 h. The RNA pellets were resuspended in 0.3 M sodium acetate and further purified with SS-phenol and chloroform. Poly(A+) RNA was then separated from the total RNA by using oligo(dT)—cellulose (Collaborative Res., Bedford, MA) column chromatography. The poly(A+) RNA was electrophoresed in a 1.2% agarose gel containing 2.2 M formaldehyde and transferred to a nylon filter (Amersham Corp.). Multiple hybridization of the poly(A+) RNA with' 2 Plabeled HPV-16 DNA, v-myc DNA or human /3-actin gene was performed as described above for DNA hybridization.
of HPV-16 DNA dimer and vector DNA pdMMT^, was examined in NHOK by transfection using the Lipofectin reagent. The cells transfected with pMHPV-16d developed two G418resistant cell colonies. Three NHOK and G418-resistant cell colonies transfected with the vector plasmid (pdMMTn^,) only were similar in their morphology and could not be subcultured
HPV-16 and oral carclrtogenesis
< CD
CO
Immortalization of normal human oral keratinocytes with type 16 human papillomavirus
No-Hee Park1*24, Byung-Moo Min\ Sheng-lin Li1, Min Zhong Huang1, Henry M.Cherick1 and Jay Doniger3 'UCLA School of Dentistry and 2UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA 90024, USA and 3Georgetown University School of Medicine, Washington, DC 20007, USA ^ o whom correspondence should be sent at the Section of Oral Biology, UCLA School of Dentistry, 43-033 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90024, USA
Introduction Human papillomavirus (HPV*) infects oral mucosa and is associated with the development of benign epithelial tumors and hyperplasia of the oral cavity, such as squamous cell papillomas, focal epithelial hyperplasia, chondyloma acuminatum, verruca vulgaris and epithelial dysplasia (1—8). HPV is also linked to certain human malignancies. This association is based on the finding that up to 90% of the cancer tissues from genital lesions contained the viral DNA (9). Of the >60 genomes of HPV, types 16 (HPV-16) and 18 (HPV-18), as well as recently isolated types 31 (HPV-31) and 33 (HPV-33), are most frequently associated with cervical cancer (10-12). They are found in up to 90% of stage 3 of cervical intra-epithelial neoplasia as well as in two-thirds of all invasive cervical cancers. A number of cell lines derived from cervix have been shown to contain both HPV DNA (13,14) and RNA (13). Furthermore, continued expression of virus-encoded proteins corresponding to the E6/E7 open reading frames (ORFs) has also been demonstrated in some of these cell lines (15,16). In a high percentage of cervical carcinomas and in cell lines derived from these cancers, HPV-16 and HPV-18 DNAs have been found to
Primary culture of normal human oral keratinocytes (NHOKs) Excised retromolar tissue from the oral cavity of a healthy male volunteer (with human subject committee approval) was washed in calcium- and magnesium-free Hanks' balanced salt solution (CMF-HBSS, GIBCO, Grand Island, NY). The tissue was incubated in CMF-HBSS containing collagenase (type II; 1.0 mg/ml, Millipore Corp., New Bedford, MA) and dispase (grade II; 2,4 mg/ml, Boenringer-Mannheim, Indianapolis, IN) to separate the epithelium from the underlying mesenchyme for 60 min at 37 °C in an atmosphere of 95% air and 5% CO}. Separated epithelial sheets were then dissociated into single cells by incubation in trypsin with agitation at 37°C for 30 min. The cells were washed with PBS, resuspended with keratinocyte growth medium (KGM; supplemented with pituitary extract, Clonetics Corp., San Diego, CA), and plated on plastic at a density of 5 x 104 cells per 28 cm2 (60 mm Petri dish).
•Abbreviations: HPV, human papillomavirus (HPV-16, type 16 HPV, etc.); ORF, open reading frame; NHOK, normal human oral keratinocyte; HOK-16A and HOK-16B, human and keratinocytes 16A and 16B; CMF-HBSS, calciumand magnesium-free Hanks' balances salt solution; KGM, keratinocyte growth medium.
Recombinant plasmid pMHPV-16d, a head-to-tail dimer of HPV-16 DNA inserted into the BamHl cloning site of the plasmid pdMMT,,^, containing G418 expression marker, was constructed as described earlier (28). The plasmid was then transfected into Escheridiia coli 294 (Genentech, South San Franciso, CA), amplified and purified with conventional methodology.
© Oxford University Press
Materials and methods
1627
Downloaded from http://carcin.oxfordjournals.org/ at Emory University on June 9, 2014
Primary human oral keratinocytes were transformed by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA, and two transformed cell lines named human oral keratinocytes-16A and -16B (HOK-16A and HOK-166) were established. While normal cells and cells transfected with vector only exhibited a limited lifespan, the HOK-16A and HOK-16B lines demonstrated immortality and altered morphology from their normal counterpart. The HOK-16A and HOK-16B lines contained ~ 40 and ~ 25 copies of intact HPV-16 DNA as integrated form per cell respectively, and both cell lines expressed several viral specific poly(A+) RNAs. Notably these cell lines also overexpressed cellular myc proto-oncogene in comparison with the normal counterpart. However, the immortalized cell lines were not able to produce tumors in nude mice, indicating that the cells are partially transformed. The HOK-16A and HOK-16B lines are, therefore, useful for investigating the multistep molecular events of oral carcinogenesis.
be integrated into cellular chromosomes, while the viral DNAs are generally retained as extra chromosomal episomes in premalignant dysplastic lesions (17-19). As in cervical cancers, HPV is also positively correlated with human oral malignancies. This relationship is explained by up to 28% of the cancer tissues from oral biopsies containing die viral DNA (20). Since the epithelium of oral mucosa and that of the female genital tract are histologically similar and they are continuously challenged by many environmental factors, close association of HPV widi the development of oral malignancies is not surprising. The availability of recombinant HPV DNA has allowed the investigation of the transforming activity of HPV in cell culture systems, which has provided supportive evidence of the transforming capacity of HPV. Morphologic transformation of C127 cells with HPV types 1 (HPV-1) and 5 (HPV-5) has been demonstrated (21). Neoplastic transformation of NTH/3T3 cells has been shown widi recombination HPV-16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full length of the HPV-16 genome (22). Similarly, transformation of human skin fibroblasts and keratinocytes was established with cloned HPV-16 and HPV-18 DNA (23-26). More recently, transformation and immortalization of normal human exocervical and cervical epithelial cells—in vivo target cells for HPV infection in genital region—was demonstrated with recombinant HPV-16 and HPV-18 DNA (26,27). Although oral keratinocytes are one of the major target cells for HPV infection and HPV-induced tumorigenesis in humans, the in vitro transforming activity of HPV in human oral keratinocytes has never been studied because of the unavailability of a culture system. In the present study, we cultured monolayers of human oral keratinocytes, transformed the cells with the introduction of the recombinant HPV-16 DNA into die cells by using liposome, and characterized the biological properties of the cells. The morphology, immortality, copy number of HPV-16 DNA per transformed cell, viral gene expression and cellular myc gene expression of die transformed cells were investigated.
N.-H.Park et al. Transfection of NHOKs Approximately 70% confluent primary NHOKs grown in 60 mm Petri dishes were transfected with pMHPV-16d or pdMMT^ by using the Ljpofectin reagent (BRL life Technologies Inc., Gaithersberg, MD). The Lipofectin reagent is a 1:1 (w/w) liposome formulation of the cationk lipid jV-[l-(2,3-dioleyloxy)propyl]-A'M/V-trirnethylaminocriloride (DOTMA) and dileoyl phsophatidylethanolamine (DOPE) in water. When mixed with DNA, the reagent interacts with DNA to form a lipid-DNA complex with complete entrapment of DNA (29). For each 60 mm dish, a mixture of 10 ^g/50 pi of pMHPV-16d (or pdMMT,,^) and 35 (ig/50 fii of Lipofectin reagent was added to culture medium, dropwise, as uniformly as possible with gentle swirling. The cells were incubated for 24 h at 37°C. The medium was then replaced with fresh KGM, and the cultures were incubated for an additional 48 h. To select cells transfected with the pMHPV-16d or pdMMTIKO plasmid, the cells were incubated in culture medium containing 70 jig/ml of G418 (GIBCO, Grand Island, NY) for 2 days, and cultured in fresh KGM for 7 additional days before subculture. Two G418-resistant cell colonies transfected with pMHPV-16d were isolated, subcultured and named HOK-16A and HOK-16B lines. Three G418-resistant cell colonies transfected with were also separated.
Fig. 1. Microscopic features of Giemsa-stained monolayers of NHOKs (A) and immortal HOK-16A (B) and HOK-16B lines ( Q . The HOK-16A and HOK-16B lines show denser growth pattern and morphological alterations. X130.
Probes and radiolabeling Linear HPV-16 DNA insert (79 kb in size) separated from the pMHPV-16d, v-myc oncogene (ATCC, Rockville, MD), and the human /3-actin gene (from Dr L.Kedes, Stanford University, Palo Alto, CA) were used as probes. They were labeled with 32 P by using the multiprime DNA labeling system (Amersham). The specific radioactivity of the labeled probes was always > 5 x 10s c.p.m./^g of DNA. In vivo tumorigenicity of H0K-16A and HOK-16B NHOK, HOK-16A and HOK-16B monolayer cultures were trypsinized, resuspended in PBS and injected s.c. into 15 athymic nude mice (nulnu; 1 x 107 cells/0.1 ml per animal; five animals per cell type) 1 day after mice had been X-irradiated (300 R). All mice were injected on the right flank and monitored twice weekly for the appearance of tumors over a period of > 3 months.
Results Proliferation pattern and morphology ofHOK-16A and H0K-16B The transforming activity of pMHPV-16d, a recombinant plasmid 1628
4.4 Fig. 2. Southern blot hybridization of high mol. wt cellular DNA (from NHOKs, H0K-16A and HOK-16B) to ["P1HPV-16 DNA after digestion with BamHI. The first three lanes represent a construction experiment in which an amount of pMHPV-16d DNA equal to 5, 25 or 125 copies per cell was rruxed with BamHI-digested NHOK DNA.
Downloaded from http://carcin.oxfordjournals.org/ at Emory University on June 9, 2014
DNA and RNA analysis High mol. wt cellular DNA from NHOK, HOK-16A and HOK-16B lines was extracted with phenol/cruoroform/isoamyl alcohol (25:24:1) and ethanol precipitated (30). To determine the presence of HPV-16 DNA and its copy number per cell if present, HPV-16 DNA [corresponding to 5, 25 and 125 copies of viral DNA per cell with BamHI-digested of NHOK DNA (10 /ig) as earner] and BamHI-digested cellular DNA (10 ^g) extracted from NHOK, HOK-16A and HOK-16B were electrophoresed in 1% agarose gel. The gel was treated with 0.25 M HCl to partially depurinate the DNA and transferred to a ntirocellulose filter (Amersham, Arlington Heights, IL) by Southern blotting (30). The nitrocellulose filter was hybridized to "P-labeled 7.9 kb total HPV-16 DNA under stringent condition (50% formamide in the presence of 10% dextran sulfate at 42°C for 16 h). The filter was washed twice in 2 x SSC buffer (1 x SSC: 150 mM NaCl plus 15 mM sodium citrate, pH 7.0) containing 0.5% SDS for 30 min at room temperature and twice in 0.1 x SSC containing 0.1 % SDS at 50°C for 30 min and exposed to Kodak SB-5 X-ray film (Eastman Kodak Co , Rochester, NY) at -70°C for 24 h. The density of the hybridized HPV-16 DNA bands was analyzed with a computing densitometer (Molecular Dynamics, Sunnyvale, CA). To determine the physical state of viral DNA in the immortalized HOK-16A and HOK-16B lines, 10 jig of high md. wt cellular DNA was digested with BamHl (B) and/or EcoRV (E) restriction enzymes. BomHI enzyme separates vector from HPV-16 sequences, while EcoRV does not digest pMHPV-16d. The fragmented DNAs were then transferred to nitrocellulose filter and hybridized to KP-labeled 7.9 kd HPV-16 DNA under stringent conditions. After washing the filter was exposed to X-ray film. To determine the expression of HPV DNA and c-myc proto-oncogene from NHOK and the immortalized cell lines, cellular poly(A + ) RNA was extracted from the cells and analyzed with Northern blot hybridization (30). Confluent NHOK, HOK-16A and HOK-16B lines were lysed with cold guanidine isothiocyanate. The lysatcs were layered onto CsCl gradient and centrifuged at 53 000 r.p.m. for 24 h. The RNA pellets were resuspended in 0.3 M sodium acetate and further purified with SS-phenol and chloroform. Poly(A+) RNA was then separated from the total RNA by using oligo(dT)—cellulose (Collaborative Res., Bedford, MA) column chromatography. The poly(A+) RNA was electrophoresed in a 1.2% agarose gel containing 2.2 M formaldehyde and transferred to a nylon filter (Amersham Corp.). Multiple hybridization of the poly(A+) RNA with' 2 Plabeled HPV-16 DNA, v-myc DNA or human /3-actin gene was performed as described above for DNA hybridization.
of HPV-16 DNA dimer and vector DNA pdMMT^, was examined in NHOK by transfection using the Lipofectin reagent. The cells transfected with pMHPV-16d developed two G418resistant cell colonies. Three NHOK and G418-resistant cell colonies transfected with the vector plasmid (pdMMTn^,) only were similar in their morphology and could not be subcultured
HPV-16 and oral carclrtogenesis
< CD
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