Detection of papillomavirus DNA in human semen.

Human papillomavirus DNA has been detected in the semen of three patients, two of whom have severe chronic wart disease. These data support the contention that sexual transmission of human papillomavirus DNA could occur via semen, a possibility suggested by epidemiological data on the sexual transmission of human papillomavirus.

Cellular transformation by human papillomavirus DNA in vitro.

Molecularly cloned DNA's of human papillomaviruses HPV-5 and HPV-l induced morphological transformation of mouse C127 cells in culture. Single-cell clones of cells transformed by papillomavirus contained multiple persistent episomal copies of the transfected DNA species and were analyzed for growth characteristics indicating malignant potential.

Correlation between phosphorylation of a 34,000-molecular-weight protein, pp60src-associated kinase activity, and tumorigenicity in transformed and revertant vole cells.

The phosphorylation of a 34,000-molecular-weight (34K) cell protein, purported to be a substrate of the avian retrovirus pp60src-associated protein kinase activity, was compared in three types of Rous sarcoma virus-infected vole cells: fully transformed cells, partial revertants which are morphologically normal in appearance but retain their tumorigenic potential, and full revertants which are similar to normal vole cells in all parameters including a lack of tumorigenicity. Although similar amounts of 34K protein are present in all three cell types, phosphorylation of the 34K protein was significantly reduced in the full revertant cell type. The reduced phosphorylation occurred at the tyrosine residue.

Functional analysis of elements affecting expression of the beta-actin gene of carp.

Regulatory regions of the beta-actin gene of the common carp (Cyprinus carpio) have been examined by linking upstream, 5'-flanking sequences and regions of the first intron to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. By analysis of the mRNA products and encoded CAT activity, we have identified four putative regions that influence expression: (i) a negative regulatory region 2,300 to 1,100 base pairs (bp) ahead of the gene; (ii) a proximal promoter element, containing the highly conserved CCAAT, CC(A/T)6GG, and TATA boxes, that is within the first 204 bp upstream of the initiation site; (iii) a negative element of 426 bp in the 5' region of the first intron; and (iv) a positive 304-bp element near the end of the first intron that contains highly conserved sequences found in all characterized beta-actin genes. The positive intron element is not a classical enhancer; it is position and orientation dependent, as has been observed in other housekeeping genes in vertebrates. Depending on the elements joined together, CAT gene expression can be modulated more than 500-fold in transfected mouse cells.

Cellular transformation by a unique isolate of human papillomavirus type 11.

Infection with human papillomavirus type 11 (HPV 11) is associated with benign epithelial proliferations and rarely with malignant and metastasizing tumors. Because of the biological diversity displayed in tissues infected with HPV 11, we have examined the capacity of various isolates of HPV 11 to transform cultured cells and compared their molecular differences by DNA sequence analysis. Five isolates of HPV 11 were examined for their ability to transform primary neonatal rat kidney epithelial cells and NIH 3T3 mouse fibroblasts in DNA transfection experiments using calcium phosphate precipitation. Included in these studies are the prototype isolate from a laryngeal papilloma (HPV 11P); HPV 11VC from a verrucous carcinoma of the penis; HPV 11Epi from the viral episomes of a primary squamous cell carcinoma; and two integrated genomes (HPV 11Int 1 and HPV 11Int 2) of the metastases. Only HPV 11VC cotransfected with the oncogene Ha-ras transformed neonatal rat kidney epithelial cells with an efficiency comparable to that of HPV 16 DNA. HPV 11VC DNA alone transformed NIH 3T3 cells. Analysis of the DNA sequence of HPV 11P and 11VC revealed 16 single nucleotide changes in the upstream regulatory region and open reading frames E1, E2, E4, and E5, five resulting in amino acid substitutions. This is the first demonstration of cellular transformation by a natural isolate HPV 11 DNA in vitro and illustrates that minimal changes in the DNA sequence of certain viruses confer oncogenicity to what are normally nontransforming viruses.

Detection of human papillomavirus DNA in invasive carcinomas of the cervix by in situ hybridization.

An examination of 27 invasive cancers of the cervix was performed using the technique of in situ hybridization using human papillomavirus DNA probes. Four tissues, previously found to harbor papillomavirus DNA by filter hybridization, were confirmed by in situ analysis. One further tissue never previously studied was also found to be positive by in situ hybridization. Overall, we found 33% of invasive cancers of the cervix to contain human papillomavirus DNA. In contrast, 55% of carcinoma in situ and severe dysplasia of the cervix were found to be positive for human papillomavirus DNA. These results confirmed that the sample population of patients in our studies have a relatively low association of human papillomavirus DNA with invasive cancers of the cervix and that in situ hybridization provides an effective complementation to filter hybridization for human papillomavirus-infected tumors.

Characterization of integrated human papillomavirus type 11 DNA in primary and metastatic tumors from a renal transplant recipient.

A primary perianal squamous cell carcinoma and two metastatic tumors from a renal transplant recipient with a previous history of condyloma acuminatum were analyzed by filter hybridization for the presence of human papillomavirus (HPV) DNA. Each of the DNA extracts from these three tissues was found to contain HPV DNA. Stringent hybridization and restriction endonuclease analysis identified this viral DNA as HPV 11 related, which largely comigrated with cellular DNA, suggesting the presence of integrated viral DNA. Each DNA extract was analyzed by two-dimensional gel electrophoresis, which separates circular and linear forms of DNA and can demonstrate linear viral DNA, which comigrated with high molecular weight linear cellular DNA, thus implying viral integration. In all three cases the vast majority of viral DNA was found to comigrate with linear DNA; in addition, a significant portion comigrated with high molecular weight cellular DNA, suggesting the presence of integrated viral DNA in these tumors. Restriction endonuclease analysis of high molecular weight cellular DNA from each of these tumors revealed identical banding patterns, indicating that the integration site in each tissue is identical and, therefore, that all three tumors most likely originated from a single clonal event. These molecular results are presented in light of the clinical history of this patient with a histologically "low grade," but biologically aggressive, squamous cell carcinoma and suggest that HPV 11 may be associated with the initiation of malignant epithelial neoplasms.

Human papillomavirus types and localization in adenocarcinoma and adenosquamous carcinoma of the uterine cervix: a study by in situ DNA hybridization.

Formalin-fixed, paraffin-embedded tissues from 108 cases of invasive carcinoma of the uterine cervix, consisting of 40 cases of adenocarcinoma, 44 cases of adenosquamous carcinoma, and, as a control, 24 cases of squamous cell carcinoma were examined for the presence of human papillomavirus (HPV) DNA by in situ hybridization of high sensitivity using tritium-labeled HPV-2, HPV-6, HPV-16, and HPV-18 DNA probes. This method detects five genome copies of homologous HPV DNA per cell. HPV DNA was detected with mixed HPV DNA probes in 17 cases (42.5%) of adenocarcinoma, 16 cases (36.4%) of adenosquamous carcinoma, and in 13 cases (54.2%) of squamous cell carcinoma. The types of HPV DNA in the HPV-positive tissues were also analyzed with each individual probe under high stringency conditions. HPV-18 DNA was detected in all but one case of the HPV DNA-positive adenocarcinoma and one-half of the HPV DNA-positive adenosquamous carcinoma. HPV-16 DNA was detected in one case of the HPV DNA-positive adenocarcinoma, one-half of the HPV DNA-positive adenosquamous carcinoma, and all cases of the HPV DNA-positive squamous cell carcinoma. HPV DNA was confined to the areas of carcinoma and squamous cervical intraepithelial neoplasia (CIN) associated with carcinoma. Among 36 cases in which CIN was associated with adenocarcinoma (9 cases), adenosquamous carcinoma (19 cases), and squamous cell carcinoma (8 cases), the same type of HPV DNA was present in the carcinoma and the associated CIN that constituted 12 cases (3 adenocarcinoma, 5 adenosquamous carcinoma, and 4 squamous cell carcinoma). Two cases (one adenocarcinoma and one adenosquamous carcinoma) contained HPV DNA in the carcinoma but not in the associated CIN. The incidence of HPV DNA did not show a significant correlation with the existence of CIN or histological differentiation of carcinoma.

Histological types of carcinoma of the uterine cervix and the detectability of human papillomavirus DNA.

Using the Southern DNA hybridization technique, tissues from 17 cases of invasive carcinoma of the uterine cervix, including nine cases of squamous cell carcinoma, four cases of adenocarcinoma, one case of adenosquamous carcinoma, and three cases of undifferentiated carcinoma, were examined for the presence of human papillomavirus (HPV) DNA. None of the studied cases had histologically confirmed association of condyloma acuminatum or cervical intraepithelial neoplasia in the vicinity. HPV DNA was detected in two of 17 cases under low stringency conditions. One lesion was undifferentiated carcinoma, and another was squamous cell carcinoma. Hybridization under high stringency conditions with a variety of HPV DNA probes indicated the presence of HPV-16 in these two lesions. The other HPV-positive lesion was adenocarcinoma, demonstrating weak hybridizations with HPV-2 and HPV-16 DNA probes only under high stringency conditions. Altogether, three of 17 cases (17.6%) contained HPV DNA. This observation contrasts to the rate of HPV DNA present in 15 of 18 cases (83.3%) of the tissues of cervical intraepithelial neoplasia. Our data suggest that HPV was not consistently detected in invasive squamous cell carcinoma, despite the frequent association of HPV with its supposed precursor lesions of cervical intraepithelial neoplasia.

Analysis of the genetic complexity and abundance classes of messenger RNA in human liver and leukemic cells.

In an effort to determine the number of genes expressed as messenger RNA in disparate human tissues we have analyzed the genetic complexity of the polyribosome-associated poly(A)-containing RNA population obtained from liver and lymphoblastic leukemic cells. This was accomplished by measuring the kinetics of hybridization of mRNA to a complementary DNA probe synthesized by avian myeloblastosis virus reverse transcriptase in vitro. The results obtained from such an analysis revealed the presence of two major abundance classes of mRNA with a total genetic complexity of approximately 10,000 diverse mRNA species in both of these cell types. Diversity of mRNA species in these unrelated human cells was studied by heterologous hybridization reactions between the cDNA of one cell type and a vast excess of poly(A)-containing mRNA from another. These types of studies indicated that extensive homology (more than 80%) exists in the mRNA sequences of disparate human cell types and suggest that the vast majority of genetic information expressed as mRNA is required for the maintenance of cellular functions common to functionally different human tissues.

Molecular cloning and characterization of a unique type of human papillomavirus from an immune deficient patient.

Several papillomas from a single patient who exhibited an unusual immune deficiency syndrome were analyzed for the presence of specific human papillomavirus (HPV) types. Preliminary analysis indicated that the HPV DNA species present in each of these tissues was quite unlike any of the previously characterized HPV types. In order to more rigorously analyze the HPV from this patient we have isolated the HPV DNA by molecularly cloning it into a bacteriophage lambda vector and have constructed a detailed restriction endonuclease map. Comparative hybridization studies using S1 nuclease analyses showed 6% or less nucleotide sequence homology of this viral DNA with HPV types 1, 2, 3, 4, 5, 6, or an HPV-11, molecularly cloned in this laboratory. Moreover, Southern blot analyses under stringent hybridization conditions revealed little, if any, hybridization to HPV types 1, 2, 4, 5, 7, 8, 10, 11, HPV-EV isolated from a patient with epidermodysplasia verruciformis (EV), or 2 previously described HPVs (HPV-P and HPV-PW) related to HPV-3. There was, however, a very weak sequence homology detected with HPV-6 and an extremely weak homology to HPV-3. No filter hybridization was observed with the recently characterized HPVs 9 or -12 to -24. These data accumulatively indicate that the HPV species from this immunosuppressed patient represents a new, hitherto unidentified HPV type.

Importance of the CArG box in regulation of beta-actin-encoding genes.

The beta-actin-encoding gene (Act) in carp is regulated by several cis-acting regulatory elements including the evolutionarily conserved CC(A/T)6GG (CArG box or serum-response element) sequences positioned in the promoter region between the CAAT and TATA boxes and in the first intron. To address the roles of the two CArG boxes on gene expression, we replaced them with linker sequences. The CArG box in the proximal promoter was not required for promoter activity in tissue-cultured cells, but was required in conjunction with a second CArG box in the first intron to give full expression in transgenic embryos. Likewise, the geometry of cis-acting transcriptional elements in the proximal promoter was more important for expression of transgenic constructs in developing embryos than in tissue-cultured fibroblasts. Mobility-shift and exonuclease mapping experiments indicated that the same or similar protein factors bind around the two CArG boxes, suggesting that interactions between the promoter and the first intron are involved in Act regulation.

HLA class I sequence-based typing.

HLA oligogenotyping has been used successfully to characterize most phenotypically undetectable variants of class II genes. Limitations inherent to the class I system have, however, complicated the application of this and other molecular approaches to HLA class I typing. We have previously shown that HLA class II polymorphism can be analyzed by a SBT approach. Here we present a class I-SBT strategy that provides complete sequence information for the two most polymorphic exons of the HLA-A, -B, and -C alleles. HLA class I SBT is based on direct sequencing of PCR-amplified HLA-A, -B, and -C cDNAs and requires a total of six cDNA -PCR-sequencing reactions (two per locus) and 13 different oligonucleotides. Each combination of oligonucleotides per reaction results in locus-specific sequence ladders and allows identification of both alleles in heterozygotes. Application of HLA-A, HLA-B, and HLA-C SBT to 26 homozygous and 32 serologically heterozygous samples has resulted in the identification of 24 novel class I nucleotide sequences encoding 17 new major histocompatibility complex class I products. An unexpected high degree of heterogeneity was found at the HLA-C locus with 14 novel sequences. Although there was a good correlation between the serologic phenotypes and SBT results, HLA-C SBT of most HLA-C serologically homozygous samples (heterozygous for HLA-A and/or -B) revealed heterozygozity (six of eight). SBT, the first molecular typing approach that has been generalized to both class I and class II genes, may be of special interest in applications demanding high sensitivity and specificity, such as in paternity testing or in the evaluation of the effects of sequence allelism in the outcome of unrelated bone marrow transplantation.

HLA class II "typing": direct sequencing of DRB, DQB, and DQA genes.

Routine clinical HLA class II typing is based largely on serological and cellular methods. These methods have many drawbacks that have led to the evaluation of molecular approaches to typing, including restriction fragment length polymorphism studies and oligotyping. We present here an alternative molecular approach, sequence-based typing (SBT), that allows direct determination of the sequences of all HLA class II polymorphic genes, thus providing the most detailed information currently possible in this regard. The data presented here using SBT are based on direct sequencing of polymerase chain reaction (PCR)-amplified DRB, DQB, and DQA cDNAs using a limited number of oligonucleotides. The oligonucleotides are designed to allow simultaneous determination of allelic sequences in any heterozygote as well as characterization of DRB isotypic complexity. Two types of amplification oligonucleotides (nonconserved and/or conserved) are used for DRB typing, which involves a maximum of four simultaneous cDNA/PCR/sequencing reactions. The first of these reactions only uses conserved oligonucleotides and is designed to detect all the different DRB transcripts present in any given heterozygote; the other three reactions use nonconserved oligonucleotides and are designed to ensure the unambiguous interpretation of the most complex DRB heterozygote combinations. Characterization of DQA1 and DQB1 sequences can be performed by using conserved oligonucleotides and only involves one reaction per locus. We have applied SBT to 43 homozygous cell lines and to 38 different heterozygote combinations that had previously been serologically typed. In all cases we were able to determine the allelic composition at DRB1, DRB3/4/5 and/or DQB1, and DQA1 loci of these cell lines and subjects; our results, analyzed by blind protocol, were consistent with the serological phenotypes. SBT can be extended to class I and class III genes and is automatable. We believe that this strategy deserves further evaluation as a possible HLA typing method.

Topical CTC-96 accelerates wart growth in rabbits infected with cottontail rabbit papillomavirus.

CTC-96, a cobalt containing complex, was tested as a putative topical therapeutic agent for the treatment of papillomavirus-induced tumors in our cottontail rabbit papillomavirus (CRPV)-rabbit model system. Following experimental infection of domestic rabbits with CRPV, CTC-96 was applied to infection sites twice daily, 5 days a week for a total of 8 weeks. Two levels of concentrations of aqueous CTC-96 were compared to placebo control-treated animals. With increasing dose of CTC-96 we observed tumors earlier, larger, and more often across eight infected sites on each animal.

Ribavirin mitigates wart growth in rabbits at early stages of infection with cottontail rabbit papillomavirus.

The challenge to develop antiviral agents effective against DNA viruses such as human papillomavirus (HPV) has been dependent on finding an animal model which mimics the human forms of the disease. We have used an existing model system for the purpose of measuring the effect of antiviral drugs on the inhibition of growth of these lesions. This was based upon domestic rabbits which efficiently grow cutaneous papillomas (warts) when infected with cottontail rabbit papillomavirus (CRPV). One agent which had shown significant success in achieving these goals was ribavirin. Ribavirin was administered intradermally shortly prior to infection at multiple sites with CRPV. Following daily injections of this drug for eight weeks, we have shown a dose-dependent response which had markedly reduced the number of warts, the time of first appearance of warts and reduced the tumor mass as compared to placebo-treated control animals. At the highest dose of ribavirin tested, 30 mg/kg/day, compared to controls, the average reduction in the number of warts was 52%, the average time of first appearance of warts was 49% longer, and the average mass of the warts was reduced by 98%. No detectable antibodies to CRPV were observed in any of the animals. The only side effects which were observed was focal alopecia, and a decrease in body growth upon prolonged treatment, both of which were completely reversible. Pharmacokinetic studies established the metabolism of ribavirin over a 24-h period of time. Ribavirin administered beginning 12 or 30 days post-infection, while not reducing the number of warts, slightly retarded the growth of warts as determined by date of first appearance of warts and mass of warts.

Human papillomavirus DNA in glandular dysplasia and microglandular hyperplasia: presumed precursors of adenocarcinoma of the uterine cervix.

Presumed precursors of adenocarcinoma of the uterine cervix were investigated with specific techniques to identify human papillomavirus (HPV) DNA. The presence of HPV DNA in 36 lesions of glandular dysplasia and 16 lesions of microglandular hyperplasia of the uterine cervix was studied by in situ hybridization using 3H-labeled HPV 16 and HPV 18 DNA probes. Only two of 36 lesions (6%) of glandular dysplasia contained HPV 18 DNA, although 64% of coexisting adenocarcinoma in situ, microinvasive adenocarcinoma, and cervical squamous intraepithelial neoplasia III lesions contained HPV 18 and/or HPV 16 DNA. Two lesions of HPV 18 DNA-positive glandular dysplasia coexisted with adenocarcinoma in situ that contained the same type of HPV DNA. None of the microglandular hyperplasia lesions contained HPV 16 DNA or HPV 18 DNA. These results suggest that, if HPV infection is an initial step toward carcinogenesis, it is unlikely that glandular dysplasia and microglandular hyperplasia are precursor lesions of adenocarcinoma of the uterine cervix. A large proportion of glandular dysplasia may represent reactive lesions of endocervical columnar epithelium. Two lesions of HPV 18 DNA-positive glandular dysplasia may represent well-differentiated components of adenocarcinoma in situ of the uterine cervix.

Human papillomavirus DNA in adenosquamous carcinoma and squamous cell carcinoma of the vulva.

The tissues from 16 cases of adenosquamous carcinoma (pseudoglandular squamous cell carcinoma or adenoacanthoma of the sweat glands of Lever) and 26 cases of invasive squamous cell carcinoma of the vulva were studied for the presence of human papillomavirus (HPV) genomes using Southern blot hybridization on fresh tissues. Types 1, 2, 3, 4, 5, 6, 16, and 18 HPV DNA probes and in situ hybridization were used on formalin-fixed paraffin sections using type 2, 6, 16, and 18 HPV DNA probes. Only one case of adenosquamous carcinoma contained an undetermined type of HPV DNA, whereas five cases of squamous cell carcinoma contained HPV DNA. Three of these five cases contained type 16, one type 6 HPV, and two an undetermined type. These results demonstrate HPV DNA associations with malignancy of the vulva that are similar to those observed elsewhere in the genital tract.

Condylomatous carcinoma of the vulva with special reference to human papillomavirus DNA.

Nine cases of condylomatous carcinoma (squamous cell carcinoma arising in condyloma acuminatum) of the vulva were studied for their clinical history, histopathology, and presence of human papillomavirus (HPV) DNA. Condylomatous carcinoma occurred primarily in an elderly population with a mean age of 70 years. There was an antecedent history of vulvar condyloma in 77%, with a median of nine months before the documentation of an invasive lesion. The disease had a good prognosis, with few recurrences and no metastasis or deaths from the disease. Human papillomavirus DNA was demonstrated to be present in 55% of these tumors by either filter or in situ hybridization techniques. Both HPV 6 and HPV 16 DNA were identified in an equal number of cases.

Epidermodysplasia verruciformis. A case associated with primary lymphatic dysplasia, depressed cell-mediated immunity, and Bowen's disease containing human papillomavirus 16 DNA.

Epidermodysplasia verruciformis is a rare, often hereditary disease characterized by a generalized cutaneous infection with human papillomavirus (HPV), depressed cell-mediated immunity, and a propensity for transformation of the warty lesions to squamous cell carcinoma on primarily sun-exposed areas of the skin. A 37-year-old man with congenital lymphatic dysplasia and a history of squamous cell carcinoma of the groin and foot was observed by us to have edema of all four extremities, numerous flat warts, and pityriasis versicolor-like papules over the trunk and arms. Condylomatous lesions were noted in the groin and a periungual verrucous nodule on the thumb. Biopsies showed the trunk and arm lesions to be verrucae and the thumb lesion to be Bowen's disease. Results of molecular hybridization studies from four lesions of the arms showed the presence of only HPV 3 DNA; HPV 16-related DNA was detected in the intraepidermal carcinoma on the thumb. Immunologic evaluation revealed anergy to routine skin testing, depressed mitogen-stimulated lymphocyte transformation, decreased B-lymphocyte count, and a severe reversal of the T-lymphocyte helper:suppressor ratio.