Presence of HPV in head and neck tumours: high prevalence in tonsillar localization.

Human papillomavirus (HPV) seems to be involved in head and neck carcinogenesis. To investigate this association, viral presence and expression were analysed by polymerase chain reaction (PCR)-based methods and correlated to tumour localization, clinical-pathological aspects, and alcohol and tobacco exposure in 65 patients. HPV DNA was found in 16 cases (24.6%); the HPV types detected were: HPV16 (10 cases), HPV 6 (3 cases) HPV 33, 35, and 58 (one case each). The tonsil was the location with the highest HPV positivity (6/8, 75%). This percentage was significantly higher than that found in tumours from any other site (P<0.01). Viral transcripts of early regions were detected in all HPV16 positive tumours. HPV status was not related to age, gender, tumour stage or grade, and use of alcohol and/or tobacco. The results suggest that HPV16 is actively involved in the genesis of a subset of head and neck cancers and that the tonsillar localization may be considered a hot spot for viral transformation.

Oligomerization of RAR and AML1 transcription factors as a novel mechanism of oncogenic activation.

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

Human papillomavirus in head and neck carcinomas: prevalence, physical status and relationship with clinical/pathological parameters.

The association of human papillomavirus (HPV) infection to head and neck squamous cell carcinomas was evaluated in 66 patients affected by tumours of the oral cavity (n = 38), the tonsil (n = 4), the pharynx (n = 2), and the larynx (n = 22). HPV DNA was detected by PCR-based assays, recognizing late and early genes. Twenty-four cases were HPV infected (36.4%), mostly by high and/or intermediate risk types. HPV 16 was integrated in 7/12 positive tumours without site-specificity. HPV infection was not related to age, gender, tumour stage, differentiation grade, and use of alcohol and/or tobacco. The findings indicate that HPV infection may be related to a proportion of head and neck carcinomas but its association is not as clear as that found in cervical cancer.

Overexpression of p53 and bcl-2 proteins and the presence of HPV infection are independent events in head and neck cancer.

The expression of p53 and bcl-2 proteins by immunohistochemistry and the identification of human papillomavirus (HPV) infection by a non-isotopic polymerase chain reaction (PCR)based method were investigated in 30 patients with head and neck cancer. Ten cases were HPV-positive (33%), mostly as double or multiple infections by high- or intermediate-risk types. Twenty-one patients were p53-positive (70%), 9/10 with HPV-positive tumours and 12/20 with HPV-negative tumours; this difference was not statistically significant. Only four cases were bcl-2-positive, irrespective of the presence of either HPV or p53. No correlation was found between these biological factors and tumour stage, differentiation grade, and alcohol or tobacco use. Our findings indicate that p53 is involved in the majority of cases, bcl-2 is rare, and high-risk HPV could play a key role, especially in tumours of tongue and tonsil. In conclusion p53 and bcl-2 protein expression and the presence of HPV infection are independent events in these malignancies.

Interactions of the CCAAT-binding trimer NF-Y with nucleosomes.

NF-Y is a sequence-specific evolutionary conserved activator binding to CCAAT boxes with high affinity and specificity. It is a trimer formed by NF-YA and two putative histone-like subunits, NF-YB and NF-YC, showing similarity to histones H2B and H2A, respectively. We investigated the relationships between NF-Y and chromatin using an Artemia franciscana chromatin assembly system with plasmids containing the Major HistoCompatibility complex class II Ea promoter. The NF-Y trimer, but not single subunits, protects the Y box in the presence of reconstituted chromatin, and it can bind the target sequence during and after assembly. Using reconstitution assays with purified chicken histones, we show that NF-Y associates with preformed nucleosomes. Translational analysis of various Ea fragments of identical length in which the CCAAT box is at different positions indicated that the lateral fragment was slightly more prone to NF-Y binding. In competition experiments, NF-Y is able to prevent formation of nucleosomes significantly. These data support the idea that NF-Y is a gene-specific activator with a built-in capacity to interface with chromatin structures.

In vitro reconstitution of Artemia satellite chromatin.

We report the characterization of an in vitro chromatin assembly system derived from Artemia embryos and its application to the study of AluI-113 satellite DNA organization in nucleosomes. The system efficiently reconstitutes chromatin templates by associating DNA, core histones, and H1. The polynucleosomal complexes show physiological spacing of repeat length 190 +/- 5 base pairs, and the internucleosomal distances are modulated by energy-using activities that contribute to the dynamics of chromatin conformation. The assembly extract was used to reconstitute tandemly repeated AluI-113 sequences. The establishment of preferred histone octamer/satellite DNA interactions was observed. In vitro, AluI-113 elements dictated the same nucleosome translational localizations as found in vivo. Specific rotational constraints seem to be the central structural requirement for nucleosome association. Satellite dinucleosomes showed decreased translational mobility compared with mononucleosomes. This could be the consequence of interactions between rotationally positioned nucleosomes separated by linker DNA of uniform length. AluI-113 DNA led to weak cooperativity of nucleosome association in the proximal flanking regions, which decreased with distance. Moreover, the structural properties of satellite chromatin can spread, thus leading to a specific organization of adjacent nucleosomes.

Concurrent HPV infection in oral and genital mucosa.

Screening for human papillomavirus (HPV) types was performed by a PCR-based assay on 29 women (mean age 34.0 years, range 21-48 years). HPV-DNA was demonstrated in 16 women (55.2%), with a detection rate of 37.9% in the oral cavity and 34.5% in the genital tract. HPV-16 was the most prevalent genotype (53.8%), followed by HPV-6, which was present in 34.6% of the positive samples. Other types were more rarely detected. Five subjects showed concurrent genital tract and oral cavity infections but HPV type-specific concordance was detected in only 3 patients. Multiple HPV infections were found in 9 of the 26 positive samples, where HPV-6 appeared frequently associated with the other types. These data confirm the occurrence of mixed HPV infections and the wide diffusion of different types of HPV in the genital mucosa and in the oral cavity; they also stress the need to utilize diagnostic methods with a wide typing capacity.

Satellite DNA from the brine shrimp Artemia affects the expression of a flanking gene in yeast.

We have previously revealed that in the brine shrimp Artemia franciscana an AluI DNA family of repeats, 113 bp in length, is the major component of the constitutive heterochromatin and that this repetitive DNA shows a stable curvature that confers a solenoidal geometry on the double helix in vitro. It was suggested that this particular structure may play a relevant role in determining the condensation of the heterochromatin. In this report we have cloned hexamers of highly-repetitive sequence (AluI-satellite DNA) in proximity to a yeast lacZ reporter gene on a plasmid. We find that the expression of the reporter gene is affected by the presence of this DNA in a dose- and orientation-dependent manner in the yeast, S. cerevisiae. We show that this effect is not dependent on under-replication or re-arrangements of the repetitive DNA in the cell but is due to decreased expression of the reporter gene. Our results indicate that the AluI-satellite DNA of Artemia per se is able to influence gene expression.

Phylogenetic study of bisexual Artemia using random amplified polymorphic DNA.

Study of polymorphisms in the eukaryotic genome is an important way to discover the evolutionary relationships between species. Artemia (Crustacea, Anostraca) offers a very interesting model for evolutionary studies. In fact the genus, distributed all over the world in hundreds of known biotopes, comprises both bisexual sibling species and parthenogenetic populations easily available from the Artemia Reference Center of Ghent. In spite of great interest in it and its extensive use in aquaculture, little is known about relationships between the different species and intraspecific populations. Recently it has been demonstrated that polymorphisms in genomic fingerprints generated by arbitrarily primed polymerase chain reaction (PCR) can distinguish between strains in many organisms. We have used this technique to estimate the phylogenetic relationships existing between 14 populations living in the American continent, in the Mediterranean area, and in China. The principal coordinate analysis (PCO) obtained from 86 random amplified polymorphic DNA (RAPD) markers indicates that the populations analyzed can be divided into homogeneous clusters representing the four known bisexual species--the American A. franciscana and A. persimilis, the Mediterranean A. salina, and the A. species from China.

Detection and typing of human papillomavirus by single hybridization.

A rapid and non-radioactive molecular hybridization test was developed which simultaneously detects and types different human papillomaviruses (HPV) DNA in fresh and paraffin-embedded clinical specimens. The method includes reverse blot hybridization between different recombinant HPV plasmids immobilized on nylon membrane and probe of cellular DNA amplified and biotin- or digoxigenin-labeled by the polymerase chain reaction (PCR). PCR protocol using consensus primers includes the mixing of Taq polymerase at high temperature (Hot-Start) and the addition of the hapten-conjugated nucleotide after the first ten cycles of amplification. The sensitivity level of this method resulted in detecting about 50 copies of HPV 16 for sample, independently of hapten used. The specificity of the typing method was also validated by more laborious and conventional analyses such as Southern-blot or PCR followed by several hybridizations with specific probes. Using this test HPV-11, -16, -18, -31 and -35 were typed in a number of samples from patients attending hospital. The method appears suitable for the handling of clinical samples in a selected population screening for type specific infections by HPV.

Topoisomerase I action on the heterochromatic DNA from the brine shrimp Artemia franciscana: studies in vivo and in vitro.

The genomes of higher eukaryotes contain various amounts of tandem repeated DNA sequences (satellite DNA) typically located in the constitutive heterochromatin, the most highly condensed region of interphase chromosomes. We have previously demonstrated that an AluI DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp Artemia franciscana. The analysis of cloned heterochromatic fragments revealed that this repetitive DNA shows a stable curvature conferring a solenoidal geometry to the double helix. In this paper we provide evidence, using the antitumour drug camptothecin, that, in vivo, topoisomerase I cleaves heterochromatin with a frequency comparable with that observed in the whole genome. The analysis of the break sites shows that the enzyme cleaves heterochromatic DNA at specific sites characterized by a degenerate consensus sequence. Moreover the enzyme-mediated breaks have, in vitro, a degenerate consensus sequence similar to, but not identical with, the in vivo one. Some of these sites are influenced by the DNA flanking the heterochromatic insert, suggesting that structural variations could modify the enzyme specificity.

Determinants of human papillomavirus types 16 and 18 infections in the lower female genital tract in an Italian population.

The prevalences of Human Papillomavirus (HPV) types 16 and 18 infection were determined by a non isotopic molecular hybridization assay on cervical scrapes from 738 women affected by gynaecological lesions different from malignancies. The correlation with known epidemiological risk factors for cervical neoplasia, such as sexual habits, smoking and pill use, was investigated. The overall HPV prevalence rate was 29.8% (220/738). Viral DNA sequences were detected in 26.9% (122/452) of morphologically normal cervices, in 75 of 224 HPV lesions (32.5%), in 1 case of 3 HSV2 lesions and in 22 of 59 dysplastic lesions (37.3%), and the frequency was increasing with the severity of CIN from 22.7% of CIN I to 57.1% of CIN III. The most relevant factor as relative risk for the presence of HPV 16/18 was the multiple lifetime sexual partners, whereas the other investigated factors were mainly associated with the clinically manifested HPV infection.

Nucleotide variation and molecular structure of the heterochromatic repetitive AluI DNA in the brine shrimp Artemia franciscana.

It has been suggested that DNA bending could play a role in the regulation of gene expression, chromosome segregation, specific recombination and/or DNA packaging. We have previously demonstrated that an AluI DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp A. franciscana. By the analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments we verified that the repetitive AluI DNA shows a stable curvature that determines a solenoidal geometry to the double helix. This particular structure could be of relevant importance in conferring the characteristic heterochromatic condensation. In this paper we evaluate how the point mutations that occurred during the evolution of the AluI sequence of A. franciscana could influence the sequence-dependent tridimensional conformation. The obtained data underline that, in spite of the high sequence mutation frequency (10%) of the repetitive DNA, the general structure of the heterochromatic DNA is not greatly influenced, but rather there is a substantial variation of the copy number of the repetitive AluI fragment. This variation could be responsible for the hypothetical function of the constitutive heterochromatin.

Purification and characterization of a proteolytic active fragment of DNA topoisomerase I from the brine shrimp Artemia franciscana (Crustacea Anostraca).

The ATP-independent type I topoisomerase from the crustacean Artemia franciscana was purified to near-homogeneity. Its activity was measured by an assay that uses the formation of an enzyme-cleaved DNA complex in the presence of the specific inhibitor camptothecin. The purification procedure is reported. Purified topoisomerase is a single-subunit enzyme with a molecular mass of 63 kDa. Immunoblot performed on the different steps of purification shows that the purified 63 kDa peptide is a proteolytic fragment of a protein with a molecular mass of 110 kDa. Similarly to the other purified eukaryotic topoisomerases, the crustacean enzyme does not require a bivalent cation for activity, but is stimulated in the presence of 10 mM-MgCl2; moreover, it can relax both negative and positive superhelical turns. The enzyme activity is strongly inhibited by the antitumour drug camptothecin. The enzyme inhibition is related to the stabilization of the cleavable complex between topoisomerase I and DNA.

Highly repetitive DNA sequence in parthenogenetic Artemia.

The study of the structural organization of the eukaryotic genome is one of the most important tools for disclosing the evolutionary relationships between species. Artemia (Crustacea, Phyllopoda) offers a very interesting model for speciation studies. The genus, distributed all over the world, comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, polyploidy, and heteroploidy). Digestion of genomic DNA of the parthenogenetic Artemia sp. from Tsing-Tao (China) with the restriction enzymes Eco RI and Alu I reveals that a highly repetitive sequence of 133 bp is present. The Eco RI fragment has been cloned and characterized by genomic organization. The distribution of the Eco RI family of repeats was also studied in several bisexual and parthenogenetic Artemia populations and compared with an Alu I repetitive fragment previously identified in Artemia franciscana.

A binding protein (p82 protein) recognizes specifically the curved heterochromatic DNA in Artemia franciscana.

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA replication, site specific recombination and DNA packaging. In Artemia franciscana (Phillopoda anostraca) cells we have revealed that an AluI DNA family of repeats, 113-bp in length, is the major component of the constitutive heterochromatin found in the species. By analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments and electrophoretic experiments we verified that the repetitive DNA shows a stable curvature that confers a solenoidal geometry to the double helix. Using the cloned monomeric fragment, as molecular probe, we describe the detection in an A. franciscana cell extract of a protein of 82 kDa (p82) that preferentially binds to heterochromatic DNA. This protein, purified of the other DNA binding proteins present in the crude cell extract, shows a greater affinity with the tandem copies of the AluI DNA fragment than with the monomer sequence. The binding of p82 protein to heterochromatic DNA is also drastically reduced in the presence of the antibiotic distamycin A, suggesting a role of the DNA curvature in the formation of the nucleoproteic complex.

Sequence-directed curvature of repetitive AluI DNA in constitutive heterochromatin of Artemia franciscana.

An Alu I family of repeated DNA sequence 113 bp in length was found to be the major component of the heterochromatin in Artemia franciscana. On the basis of the analysis of cloned oligomeric (monomer to examer) heterchromatic fragments we predicted that the sequence could produce a stable curvature in chromosomal DNA. This prediction was confirmed by polyacrylamide gel electrophoresis analysis and by electron microscope observations. The anomalous mobility of these fragments is reversed when the DNA samples are electrophoresed in the presence of distamycin A. Moreover treatment of living Artemia with this drug produces visible decondensation of heterochromatic masses in the interphase nuclei.

Analysis with monoclonal antibodies of the molecular and cellular heterogeneity of human high molecular weight melanoma associated antigen.

Monoclonal antibodies (MoAbs) 225.28, 657.9, and 902.5 recognizing distinct epitopes of the human high molecular weight melanoma associated antigen (HMW-MAA) were used to investigate the molecular and cellular heterogeneity of the HMW-MAA synthesized by human melanoma cells. Sequential immunodepletion and immunoprecipitation experiments showed that not all HMW-MAA molecules synthesized by a melanoma cell line express the antigenic determinants recognized by the three monoclonal antibodies. The majority of the HMW-MAA molecules expressed the epitope defined by MoAb 657.9 since this monoclonal antibody depleted the melanoma cell lysate of all antigen molecules recognized by the other two monoclonal antibodies. Depletion with MoAb 902.5 resulted in the removal of a large proportion of the HMW-MAA molecules precipitated by MoAb 657.9. The MoAb 225.28 depleted the cell lysate of only a fraction of the HMW-MAA molecules recognized by MoAb 657.9 and 902.5. Two-dimensional gel electrophoresis and peptide mapping analysis did not detect any significant difference among the HMW-MAA immunoprecipitated by the three monoclonal antibodies. The heterogeneity of the epitopes recognized by the three monoclonal antibodies is, at least partly, due to glycosylation of the antigen molecule, since treatment of melanoma cells with glycosidases differentially affects their ability to bind the three anti-HMW-MAA monoclonal antibodies. Fluorescent activated cell sorting analysis of the melanoma cells showed that the heterogeneity exhibited by the HMW-MAA is not due to the presence of different cell clones in the culture but reflects a differential distribution of epitopes on the HMW-MAA expressed on the surface of individual cells. Immunohistochemical staining of surgically removed benign and malignant lesions of melanocytic origin, of normal tissues, and of malignant lesions has shown a differential tissue distribution of the determinants recognized by the three monoclonal antibodies. Staining of melanoma cell lines and of surgically removed melanoma lesions with combinations of the three monoclonal antibodies did not cause any significant change of the percentage of stained cells but markedly increased the intensity of staining. These results indicate that combinations of monoclonal antibodies to distinct determinants of HMW-MAA can markedly increase the sensitivity of immunohistochemical techniques to detect melanoma cells.

Yeast DNA polymerase--DNA primase complex; cloning of PRI 1, a single essential gene related to DNA primase activity.

The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated. The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library. Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity. The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number. Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function.