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.

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.

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.

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.

Human papillomavirus heterogeneity in 36 renal transplant recipients.

Immunosuppressed patients such as renal transplant recipients are prone to increased incidence of wart disease. We examined 48 tissue specimens from 36 renal transplant recipients using human papillomaviruses (HPVs) 1, 2, 3, 4, 5, and 6 in filter hybridization under stringent conditions. The results showed that 90% of the samples contained HPV DNA. Of these 43 positive samples, we found HPV-1 in 2%, HPV-2 in 56%, HPV-3 in 19%, HPV-4 in 47%, HPV-5 in 9%, and HPV-6 in 5%. In several cases, more than one type of HPV DNA was observed. In a few of these cases, the clinical appearance of the lesions differed from what might have been expected, such as those lesions containing HPV-3- or HPV-5-related DNAs.

Presence of human papillomavirus in verrucous carcinoma (Ackerman) of the vagina. Immunocytochemical, ultrastructural, and DNA hybridization studies.

Human papillomavirus (HPV) genomes were identified in two cases of verrucous carcinoma of the vagina, using Southern blot DNA hybridization under low-stringency conditions. Type (group) 6 HPV DNA (HPV-6) was identified, using molecularly cloned HPV-1 through HPV-6 DNA probes under high-stringency conditions in both cases. In addition, DNA extract in one case hybridized with HPV-1, HPV-3, and HPV-4 DNA probes. No HPV structural proteins were demonstrated in either case by immunocytochemical tests, using HPV antibodies. In one case viruslike intranuclear particles were observed by transmission electron microscopy. These two cases suggest a strong associative relationship between HPV and verrucous carcinoma (Ackerman) of the lower part of the genital tract.

Regulation of HLA class I transcription in T cells.

Cortical thymocytes express a very low level of HLA class I Ag on their cell surface. As these cells mature to medullary thymocytes and peripheral T cells, HLA class I expression increases to a high level. Leukemic T cell lines arrested at various stages of development were used to examine the regulation of HLA class I expression. Nuclear runoff analysis demonstrated that expression differences in these cell lines were the result of a difference in transcription rates. Electrophoretic mobility shift analysis with the upstream regulatory region of HLA-A2 as a probe for HLA class I-specific DNA binding proteins indicated that DNA binding activity in the cell extracts paralleled the transcriptional activity of the cells. The predominant DNA binding activity in these T cell lines was mapped, by DNase I protection analysis, to a conserved inverted repeat of the MHC class I enhancer. Thus HLA class I expression in these stage-specific T cell lines correlates with the amount of this enhancer binding activity in these cells suggesting that this commonly found DNA binding factor may, in this system, act to regulate HLA class I expression in a very specific manner.

CArG, CCAAT, and CCAAT-like protein binding sites in avian retrovirus long terminal repeat enhancers.

A strong enhancer element is located within the long terminal repeats (LTRs) of exogenous, oncogenic avian retroviruses, such as Rous sarcoma virus (RSV) and the avian leukosis viruses. The LTRs of a second class of avian retroviruses, the endogenous viruses (evs), lack detectable enhancer function, a property that correlates with major sequence differences between the LTRs of these two virus groups. Despite this lack of independent enhancer activity, we previously identified sequences in ev LTRs that were able to functionally replace essential enhancer domains from the RSV enhancer with which they share limited sequence similarity. To identify candidate enhancer domains in ev LTRs that are functionally equivalent to those in RSV LTRs, we analyzed and compared ev and RSV LTR-specific DNA-protein interactions. Using this approach, we identified two candidate enhancer domains and one deficiency in ev LTRs. One of the proposed ev enhancer domains was identified as a CArG box, a motif also found upstream of several muscle-specific genes, and as the core sequence of the c-fos serum response element. The RSV LTR contains two CArG motifs, one at a previously identified site and one identified in this report at the same relative location as the ev CArG motif. A second factor binding site that interacts with a heat-stable protein was also identified in ev LTRs and, contrary to previous suggestions, appears to be different from previously described exogenous virus enhancer binding proteins. Finally, a deficiency in factor binding was found within the one inverted CCAAT box in ev LTRs, affirming the importance of sequences that flank CCAAT motifs in factor binding and providing a candidate defect in the ev enhancer.

Blackjack, a novel protein associated with microtubules in embryonic neurons.

Microtubule-associated proteins can influence the organization, stability and dynamics of microtubules. We characterize a novel protein that associates with microtubules as assessed by immunofluorescence, immunoelectron microscopy, and co-sedimentation. The protein is expressed heavily in embryonic neurons and, to a lesser extent, in epithelial and mesodermal cells. The cDNA sequence predicts a protein of 1,547 amino acids and approximately 170 kDa. Immunoblot of embryo lysate demonstrates bands of approximately 240 and 260 kDa. The predicted amino acid sequence contains 77 potential serine/threonine phosphorylation sites. A distinctive feature is a predicted alpha-helical central domain comprising 21 identical repeats of an 11 amino acid sequence (PLEELRKDAAE). The protein is thermostable and has two major charge-domains: the amino-terminal 80% has an estimated pI of 4.0 and the carboxy-terminal 20%, a pI of 12.2. The protein shares several general biochemical and molecular features of MAPs, but its sequence is not similar to that of any described MAP.

Expression of epithelial alkaline phosphatase in segmentally iterated bands during grasshopper limb morphogenesis.

Although the study of rostral-caudal segmentation of the insect body has been a rich source of information about embryonic pattern formation, relatively little is known of the process of proximal-distal segmentation of insect appendages. Here we demonstrate that during the period of limb segmentation, five segmentally iterated, sharply demarcated bands of cell surface alkaline phosphatase activity are expressed in embryonic grasshopper limbs. These bands span each intersegmental boundary in the limb as well as one boundary within the tarsus. Within appendages, expression is restricted to epithelial cells, where activity is present on both apical and basolateral surfaces. This epithelial alkaline phosphatase remains active at neutral pH, is insensitive to levamisole inhibition, and is strongly inhibited by nucleoside monophosphates. Treatment of embryos with phosphatidylinositol-specific phospholipase C releases almost all visible chromogenic activity, indicating that the epithelial alkaline phosphatase is anchored to the plasma membrane by glycosyl-phosphatidylinositol. When material released by phosphatidylinositol-specific phospholipase C is separated on native polyacrylamide gels, a single broad band of enzymatic activity is detected following incubation with substrate. A polyclonal antiserum raised against a 55 x 10(3) M(r) alkaline phosphatase from shrimp recognizes a single band of 56 x 10(3) M(r) on immunoblots of grasshopper membrane proteins. The spatially restricted expression of epithelial alkaline phosphatase suggests that it may be involved in epithelial cell rearrangements or shape changes associated with limb segmentation and morphogenesis. It also may contribute to definition of axon routes in the limb, since pioneer afferent growth cones turn at, and migrate along, the edge of one alkaline phosphatase-expressing epithelial domain.

Molecular cloning and nucleotide sequence analysis of several naturally occurring HPV-5 deletion mutant genomes.

Three deletion mutants of naturally occurring human papillomavirus type 5 (HPV-5) were molecularly cloned into phage vectors. The nature of these deletions was characterized initially by restriction endonuclease mapping and electron microscopic heteroduplex analysis and ultimately by nucleotide sequence analysis. The sizes of the deletions are 353, 1329, 1571, and 2267 bp and map to the late gene region of the HPV-5 genome. The 80 nucleotides immediately adjacent to the deletions exhibit no significant detectable sequence homologies or symmetries and therefore were probably not formed by the sequence-dependent events of homologous recombination or site-specific recombination.

Nucleotide sequence and genome organization of human papillomavirus type 5.

Human papillomavirus (HPV) type 5 is associated with benign and malignant lesions of the disease epidermodysplasia verruciformis (EV). Because of the strong correlation between the presence of HPV-5 and malignant progression in these patients, we have elucidated the nucleotide sequence of the HPV-5 genome. The size of the HPV-5 genome is 7746 nucleotides and its organization is similar to that of other papillomaviruses. The HPV-5 genome exhibits extensive sequence homology with another EV-associated papillomavirus, HPV-8, although HPV-5 appears to contain at least one additional open reading frame.

Human papillomavirus type 27: detection of a novel human papillomavirus in common warts of a renal transplant recipient.

The cloning and partial characterization of the genome of human papillomavirus type 27 (HPV-27) is described. Hybridization analyses reveal that this is a new HPV type, with the strongest homology to HPV-2.

A survey of human cancers for human papillomavirus DNA by filter hybridization.

We examined 217 tissue samples of various human malignancies for the presence of human papillomavirus (HPV) DNA using low-stringency filter hybridization techniques. These techniques were sufficiently sensitive for crosshybridization of the HPV DNA probes to all the known types of papillomavirus DNAs, both human and animal. Approximately 2% of the cancers analyzed contained HPV DNA. These included carcinomas of the lung, cecum, tongue, and neck. Three of four cancers contained HPV-16-related nucleotide sequences. Thus, in addition to previous data demonstrating the association of HPV DNA with certain cancers of the skin and genital tract, data is presented that indicates that several additional human cancers also contain HPV-related nucleotide sequences.

Identification of human papillomavirus DNA in cervical and vaginal intraepithelial neoplasia with molecularly cloned virus-specific DNA probes.

The presence of human papillomavirus (HPV) DNA was identified in the tissues of cervical and vaginal intraepithelial neoplasia by Southern blot DNA hybridization under conditions of low stringency. The specific types of HPV present in the tissues were identified by using molecularly cloned types 1 through 6 (HPV-1 through HPV-6) HPV DNA probes under high-stringency conditions. All tissues of cervical and vaginal intraepithelial neoplasia analyzed contained HPV genomes. Fifteen of 19 samples (79%) contained HPV-6 DNA, and 10 of 19 samples (53%) HPV-3 DNA. Hybridization with HPV-1, HPV-2, HPV-4, and HPV-5 DNAs was also observed in several of the samples. Four of the samples did not hybridize with any of the probes tested (HPV-1 through HPV-6); yet, all showed hybridization with an HPV-EV DNA (a type 3-related DNA) probe under low-stringency conditions, indicating the presence of HPV types other than those belonging to HPV-1 through HPV-6.

Heterologous expression of alpha 1-integrin cDNA generates variable ligand specificities and alterations in cell shape.

Integrins can mediate a diverse variety of functions that are regulated by unknown mechanisms. Integrin alpha 1 beta 1 can serve as a receptor for laminin-1 and collagen in certain cell types, but is a receptor for only collagen in others. To examine the molecular basis of this difference in specificity, three cell types were transfected with cDNA for the rat alpha 1 subunit. Following transfection with rat alpha 1, pluripotential hematopoietic human K562 cells exhibited alpha 1 beta 1-dependent attachment to collagen IV, but not laminin-1, unless activating antibody TS2/16 was added. The attachment to collagen IV stimulated the elaboration of a spread morphology resembling a differentiated megakarocyte with extensive processes which were absent in response to all other substrates. When MRC-5 cells, a human fibroblastic cell, or RD cells, a human rhabdomyosarcoma line, were transfected with the identical alpha 1-integrin construct, rat alpha 1 beta 1-dependent attachment to both collagen IV and laminin-1 was seen. Therefore differences in ligand specificity can be generated by translation of an identical integrin alpha 1 beta 1 mRNA in different cell types. Despite differences in ligand binding, alpha 1 cDNA-transfected K562 and RD cells express an alpha 1 subunit that appears to be antigenically and electrophoretically similar. Small differences in glycosylation were apparent, and correlated with changes in ligand specificity. Together these results show for the first time that identical cDNAs, absent activating antibodies or other manipulations, can change ligand selectivity and better establish the importance of cellular context in determining integrin function. Moreover they show that select integrins can shift the differentiated state of pluripotential cells.

Detection of human papillomavirus DNA in anogenital neoplasias.

The presence of papillomaviruses in epithelial-derived cancers from several animal species has led to the speculation that these viruses may also have a pathogenic role in the development of certain human carcinomas, particularly those associated with the anogenital tract. Recently, human papillomavirus (HPV) DNA has been detected in epithelial-derived cancers, both cutaneous and metastatic, from patients exhibiting the rare, chronic flat wart disease, epidermodysplasia verruciformis (EV). Except for patients exhibiting this chronic wart syndrome, the association of HPV genomes with human epithelial cancers has not been demonstrated. In an attempt to delineate the association and possible involvement of papillomaviruses with human anogenital carcinomas, we have begun an analysis of these cancers for the presence of HPV-specific nucleotide sequences by using highly sensitive hybridization procedures capable of detecting distantly related papillomaviruses at low copy number. Here we demonstrate the presence of HPV DNA in several types of anogenital tumours: Bowenoid papulosis, carcinoma in situ, and verrucous carcinoma. These data indicate that HPV can be detected in several types of premalignant and malignant tumours, supporting the contention that this group of viruses may be involved in the development of certain types of human epithelial-derived cancers.