[Flow cytometric bromodeoxyuridine (BrdU)/DNA analysis--study with urogenital tumors].

We performed the simultaneous flow cytometric bromodeoxyuridine (BrdU)/DNA analysis in combination with in vitro BrdU labeling method using seven human urogenital tumors. The DNA content and incorporated BrdU content of each tumor cell was analyzed quantitatively using this flow cytometric method. The cell kinetics of each cell line of heterogeneous tumor could be analyzed by this combined method. In the near future, by establishing a procedure decreasing non-specific staining of cells, the development of this flow cytometric two-color analysis in clinical fields is expected.

[Four putative neuropeptides concentrations in the human urogenital tract. Comparison of the neuropeptides concentration between malignant and benign tissues].

In order to evaluate a possible role of several peptides in the human urogenital tract, peptide concentrations in urogenital tissues collected from surgery were measured using specific radioimmunoassay. The specimens were extracted in boiling 0.5M acetic acid, and these extracts were utilized to measure neuropeptide concentrations, i.e., neuropeptide Y(NPY), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and peptide 7B2. The highest concentrations of NPY were found in seminal vesicle (145 +/- 42pmol/g) and vas deference (104 +/- 26pmol/g). There was no significant difference in NPY concentration between malignant and non-malignant tissues (prostate and urinary bladder). High concentrations of VIP were also observed in several urogenital tissues (seminal vesicle, vas deference and urethra). VIP concentrations in prostatic cancer and carcinoma of urinary bladder seemed to be reduced, though no significant difference could be found in each corresponding tissue. Pituitary peptide 7B2 was found to be present in the human urogenital tract in relatively low concentrations. A significant difference was observed in CGRP concentration between carcinoma of urinary bladder and adjacent normal vesicular tissues (p less than 0.05). These four peptide immunoreactivities were further characterized by gel permeation or high performance liquid chromatography. Each main immunoreactivity in urogenital extracts seemed to correspond to each synthetic standard or pituitary extracts (in case of 7B2). These results demonstrated that pituitary peptide 7B2 was shown to be present in the human urogenital tract and that the distribution patterns of these peptides might correlate to their pathophysiological role in the urogenital tract. Furthermore, the absence of CGRP immunoreactivity in carcinoma of urinary bladder may be useful for additional diagnostic information.

Sensitive detection of nucleic acids and protein of human papillomavirus type 6 in respiratory and genital tract papillomata.

We have developed a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in biopsy specimens of genital and respiratory tract lesions by using in situ hybridization and immunoperoxidase assays on sections of plastic-embedded tissue. This modified in situ hybridization technique, using ultrathin sections and strand-specific 3H-labelled riboprobes, offers the advantages of superior morphological preservation and detection of viral genomes at low copy number with good resolution. This modified immunocytochemistry provides better sensitivity when compared to previous methods using paraffin-embedded materials. In respiratory tract lesions, immunoperoxidase assay detected only a few capsid antigen positive cells, while in the genital tract lesions, there were more capsid antigen positive cells. Southern transfer analyses and in situ hybridizations demonstrated the presence of more viral nucleic acids in genital tract papillomata than respiratory tract papillomata. Epithelial cells throughout the papillomata were infected by HPV-6 as evidenced by positive hybridization, with more viral DNA present in superficial cells. Our results suggest that genital tract epithelium is more permissive for HPV-6 replication than respiratory tract epithelium. Using stand-specific probes synthesized from subgenomic fragments of the HPV-6 genome in conjunction with nuclease digestions, we were able to demonstrate that HPV-6 transcripts specific to open reading frames (ORFs) E6, E7, E1, L1, and L2 occur in maturing superficial cells. In contrast, transcripts specific to ORFs E1, E2, E4, E5a, and E5b could be detected throughout the whole of the epithelium with more signals noted at the basal cell areas. In addition, the distribution of HPV-6 nucleic acids and protein in a carcinoma in situ of the larynx was analyzed. In comparison to benign respiratory tract papillomata, more viral DNA was found in the malignant lesion, but the pattern and distribution of transcription and capsid antigen was similar.

Flow cytometric analysis of relative mean DNA content of urogenital cancer cells in fresh and paraffin-embedded materials.

The relative mean DNA content calculation was performed by flow cytometry on single cell suspensions prepared from fresh and paraffin-embedded specimens of 10 patients with surgically resected urogenital cancer. Samples were processed by a modified method of Hedley et al. including two hours of pepsinizing time, ribonuclease digestion, and propidium iodide staining. The mean DNA content which is a quantitative description of flow cytometric characteristics was significantly correlated between the fresh and paraffin-embedded materials (n = 10, r = 0.869, p less than 0.01). This method allows for the objective, retrospective analysis of DNA content in relation to diagnosis and prognosis of urogenital cancer.

The effect of cell extracts of urological tumours on adenovirus mutants.

Various tumours of the urogenital system were studied whether their cells contain gene products characteristic of functioning adenovirus genes. Complementation studies were made by using tumour cell extracts and two temperature-sensitive adenovirus mutants at restrictive and permissive temperatures. The procedure was applied in in vitro HEp-2 cell culture. It was found that cell extracts of bladder tumour, renal tumour, malignant bladder papilloma and of penis cancer are capable of complementing adenovirus mutants at restrictive temperature, i.e. they induce the reproduction of virus mutants being otherwise incapable of replication. Thus, functioning adenovirus genes are present in the tumour cells. It seems that virus genes together with hormonal changes activate the cell protooncogenes giving rise to and maintaining tumours.

Intermediate filament proteins as tissue specific markers in normal and malignant urological tissues.

Immunocytochemical techniques have become valuable tools in many fields of clinical pathology and medical research. Especially the development of highly specific (monoclonal) antibodies to a large variety of tissue antigens has in recent years led to the establishment of sensitive tissue markers. One of the most promising types of tissue specific markers so far is represented by the intermediate filament proteins. Since the findings of this rapidly expanding field are also being applied in urology, we have reviewed the current data in order to describe the new insights in tumor biology and histogenesis, as well as their application in diagnostic pathology.

Gangliorhabdomyosarcoma: a histopathological and immunohistochemical study of three cases.

A histopathological and immunoperoxidase study on three cases of genitourinary gangliorhabdomyosarcoma using a spectrum of conventional staining methods and antibodies against myoglobin, neuron-specific enolase and S-100 protein is presented. The results of the study have shown that differentiated myoblasts, ganglion cells and Schwann cells reacted positively with the particular antisera, but the majority of undifferentiated cells were negative. From the immunopathology results it was not possible to determine whether the undifferentiated cells were precursors of neural cells or myoblasts; the histological appearance resembled that of mesenchymal cells commonly seen in rhabdomyosarcomas. Theories concerning the origin of these tumours from neural crest ectomesenchyme or from neural crest and somitic mesenchyme are considered. Further study is needed to establish their histogenesis.

[Study on heterotransplantation of malignant urogenital tumors in nude mice: results of transplantation and the characteristics of the explants].

Since 1976, we have transplanted 82 urological neoplasms into nude mice, 46 of which (56%) took. Thirty five of them (43% of the total tumors) are being serially transplanted. This rate of success seems to be better than that obtained at other institutes for both neoplasms of urogenital as well as other tissue origin. The explants basically retained the original characteristics of the native tumors not only in histological pattern but also in tumor markers, even after a long term period of heterotransplantation. However, the histological features of some tumor lines seemed to be reduced. A certain cell population was lost during repeated transplantations. Such a clonal selection may have resulted from the outgrowth of the cell population capable of adapting to the transplanted environment. Nevertheless heterotransplantation experiments in nude mice are one of the most valuable tools in various cancer research including that in the urological field since a rather high percentage of urologic malignancies take while retaining their original characteristics for a long time.

The contribution of immunohistochemistry to the diagnosis of neuroendocrine tumors.

The use of specific secretory products, hormones, and neuroregulators as diagnostic tools for neuroendocrine tumors is illustrated. Results of extensive immunohistochemical and cytologic investigations are discussed in the light of other pathologic and clinical findings to serve as a basis for tumor classification and as a help in prognostic evaluation. Endocrine tumors of the pancreas, gut, lung and urogenital tract are dealt in some detail.

Isolation of a human papillomavirus from a patient with epidermodysplasia verruciformis: presence of related viral DNA genomes in human urogenital tumors.

The DNA genome of a human papillomavirus (HPV), tentatively designated HPV-EV, was molecularly cloned from hand to leg lesions of a patient with epidermodysplasia verruciformis, a chronic skin disease associated with a 30% risk of developing cancer. Using stringent hybridization conditions, we observed less than 5% homology between HPV-EV and the cloned genomes of HPV-1, HPV-4, HPV-5, and HPV-5a. HPV-EV DNA showed approximately 6% homology with HPV-2 and 36% homology with HPV-3. These data suggest that HPV-EV is partially related to HPV-3. Using 32P-labeled cloned HPV-EV as probe in Southern blot hybridization experiments, we detected HPV-EV-related DNA in the carcinoma in situ (Bowenoid lesion) of the vulva of the patient from which HPV-EV was isolated. HPV-EV-related DNA was detected in 2 of 10 vulva carcinomas and in 2 of 31 cervical carcinomas. Related DNA sequences were found in papillomas from each of two patients with condyloma acuminata (anogenital warts), which is of interest considering that condylomas have been reported to convert occasionally to carcinomas. The positive vulva DNAs were also probed with other cloned HPV DNAs: HPV-1, HPV-4, and HPV-5a-related sequences were not detected; HPV-3 and HPV-2 DNA probes detected strong and weak DNA bands, respectively, of the same size as found with HPV-EV. The HPV DNA sequences were present in the positive tumors mainly as free viral DNA molecules; no evidence for integration into cellular DNA was found. The emerging biological picture with papillomaviruses is that cells transformed by these viruses are maintained in a transformed state by free episomal genomes. Thus, our findings are consistent with the idea, but by no means establish, that HPVs play a role in human cancer by a similar mechanism.

[Radioimmunoassay of SP1 in obstetrics and gynecological oncology]. / Dosaggio radioimmunologico della SP1 nella pratica ostetrica e nella oncologia ginecologica.

The authors by means of radioimmunoassay analyze SP1 values in selected obstetric and oncological samples. Seven amniotic fluid samples showed various degrees of positivity, but not strictly correlated to the gestational age (previous analysis by immunodiffusion were always negative). SP1 concentrations in ectopic pregnancy confermed similar investigations by other authors. In the oncological field only one serum was highly positive (440 ng/ml); negativity of others could be due to the long time of storage. The SP1 was assayed using a sperimental kit supplied by Boehringwerke A.G. (Marburg, W.G..). Assay procedure: preparation of a standard curve (7 - 440 ng/ml) from which the unknown SP1 content was determinated. Reaction time: 16 - 24 h/20 degrees C (first incubation) and 0.5 - 4 h/20 degrees C (second incubation).

Analysis of human genital warts (condylomata acuminata) and other genital tumors for human papillomavirus type 6 DNA.

32P-labelled cloned HPV 6 DNA was used as probe to analyze human genital tumors for DNA sequences homologous to HPV 6 DNA. Ninety three percent of all condylomata acuminata (41 out of 44) were found to harbor HPV 6 DNA. Of the remaining three, one contained HPV 1 DNA. No papillomavirus DNA was identified in the two other tumors. All three invasively growing giant condylomata acuminata (Buschke-Löwenstein tumors) investigated also contained HPV 6 DNA. Two out of six atypical condylomata of the cervix hybridized with HPV 6 DNA under stringent conditions, one only under conditions of low stringency. All DNA preparations from malignant tumors studies (54 cervical carcinomas, 10 penile carcinomas, two vulvar carcinomas) failed to anneal with HPV 6 DNA, even under conditions of low stringency. Although all HPV 6-positive condylomata acuminata analyzed in this study revealed HPV 6 DNA of regular molecular weight (5.1 x 10(6)), two of the Buschke-Löwenstein tumors, as well as one of the two positive atypical condylomata of the cervix, contained HPV 6 DNA with a remarkable size classes occurred in a supercoiled form without evidence for integration into host cell DNA.

Molecular evidence of viral-like biochemical activities in human genitourinary malignancies.

We have demonstrated previously that core structures of urine samples from patients with genitourinary malignancies contain ribonucleic acid-directed deoxyribonucleic acid polymerase and a high molecular weight ribonucleic acid. If these particles originated from the existing genitourinary malignancies then the malignancies should contain similar characteristics. We examined 13 prostatic carcinomas, 4 bladder carcinomas, 1 urethral carcinoma and 1 hypernephroma. Positive reactions were noted in 10 of the 13 prostatic carcinomas (77 per cent), all 4 bladder carcinomas, the 1 urethral carcinoma and the hypernephroma with the simultaneous detection assay. The control samples consisted of 7 tissues of benign prostatic hypertrophy, and tissue from 2 normal bladders and 1 normal kidney. None of these tissues showed a positive response. Tritium labeled deoxyribonucleic acid probes synthesized from the malignant tissues hybridized to the polysomal ribonucleic acids but not to the corresponding normal tissues. Particles derived from the probes have a density of 1,1620 in sucrose gradient. No sequence homology could be demonstrated with various known oncogenic ribonucleic acid viruses nor with malignancies arising from other organs.

Analysis of human cancer DNA's for DNA sequence of human adenovirus serotypes 3, 7, 11, 14, 16, and 21 in group B1.

We have investigated whether Group B human adenoviruses (Ad) (Ad3, Ad7, Ad11, Ad14, Ad16, and Ad21), which are widespread in the human population and are tumorigenic in hamsters, may play a role in human cancer. Hybridization of Ad7-radiolabeled DNA with DNA's from an Ad7-induced primary hamster tumor and from two cell lines (5728 and Ad7 P-cell) established from Ad7-induced hamster tumors indicated multiple copies per cell of 17, 30 to 36, and 20%, respectively of the Ad7 genome. Thus, cells transformed by Group B Ads resemble cells transformed by Group C and Group A Ad's in that they retain multiple copies of a variable fraction of the viral genome. These model studies suggest that possible Group B Ad-induced human cancer cells should contain one or more copies of virus DNA per cell. Therefore, we assayed human cancer DNA's for Ad sequences, by highly sensitive "saturation-hybridization" reactions with Ad7 or Ad11 DNA (4 X 10(6) to 2.1 x 10(8) cpm/microgram). We concentrated on cancers of the respiratory and digestive systems, because these systems are the most common sites of infection by Group B Ad's. In 8 independent experiments, no Ad7 sequences were detected in DNA's from 16 normal lung tissues, 18 normal tissues of the digestive system, 34 cancers of the respiratory system, 19 cancers of the digestive system, 11 cancers of the urinary system, 5 cancers of the genital system, 3 cancers of the breast, and 6 Hodgkin's lymphomas. Reconstruction controls with added Ad7 DNA indicated that about 0.05 to 0.1 copy of Ad7 DNA per cell should be detected. Ad11 is strongly implicated as a cause of acute hemorrhagic cystitis. In two independent experiments, no Ad11 sequences were detected in DNA's from 9 carcinomas of bladder, 10 carcinomas of prostate, 24 carcinomas of kidney, 3 hypernephromas, 3 Wilms' tumors, or 2 normal kidneys. Reconstruction experiments indicated that the cancer DNA assays had a sensitivity of 0.05 to 0.1 copy of Ad11 DNA per cell. The DNA's of Group B Ad's are greater than 85% homologous by hybridization; thus, these results are applicable to all Group B serotypes. Our data provide evidence (but not formal proof) that none of the human cancers that we analyzed were induced by Group B Ad's. These tumors represent about 50% of the tumors that affect humans. The possible involvement of Group B Ad's in other less common forms of human cancers is under investigation in our laboratory.