Human papillomaviruses in cervical cancer I. HPV-16 and 18 predominate in the Greek population.

Human papillomaviruses (HPV) and their role in carcinogenesis have been the subject of extensive investigation Specific types of HPV have been associated with cervical carcinoma HPV 16 and 18 are mainly associated with malignant progression and considered "high risk" viruses Using Southern blot analysis and in situ hybridization we investigated the presence of papilloma viruses in cervical carcinoma patients as well as appropriate controls. The results presented here support the aetiological role of HPV 16 and 18 in cervical carcinoma and demonstrate the prevalence of these viruses in Greek women. The role of viruses in carcinogenesis in well established in almost all species from fishes, to birds, to mammals. Although not well circumstantiated, viruses probably play as-great a role in human cancer as in other species. The role of human papillomaviruses (HPV) not only in benign proliferations, but also in a number of malignancies has long been postulated (1,2). Presently over 20 HPV types have been identified and there is evidence now associating specific types with certain human anogenital cancers, notably cervical cancer (3,4). Advance neoplasias such as squamous cell carcinomas are associated with types, 16,18 and 31, with type 16 prevailing in these lesions (5,6). In this paper we shall present evidence which extends and confirms that previously reported on the prevalence of HPV 16 and 18 in Greek women.

Human papillomaviruses in cervical cancer: II. Application of PCR and in situ hybridization and HPV significance.

Human papillomaviruses especially type 16 and 18 are associated with cervical cancer. These viruses encode transforming proteins which are capable of binding and form complexes with p53 protein. Tumour-tissue samples from women with cervical cancer as well as normal controls were analysed for the presence of HPV and the expression of p53 by in situ hybridization and PCR analysis. The results showed that HPV 16 and 18 predominated in all pathological conditions and that p53 expression was related to the presence of certain types of HPV. The significance of HPV in cervical carcinoma is well demonstrated.

Nasopharyngeal carcinoma: Epstein-Barr Virus significance.

Thirty-one cases of NPC were investigated by in situ hybridization, PCR analysis and serology to assess the presence of EBV and to interrelate these findings. Statistical analysis showed a significant correlation between the probability of detecting EBV and NPC. There was no significant association between in situ hybridization, PCR and serology. Tumor size correlated with EBNA, but no correlation between smoking and NPC was found.

Heat turn-over during hyperthermic peritoneal perfusion. An experimental study.

In order to study heat turnover, 3 experimental models of chemotherapeutic hyperthermic intraperitoneal perfusion (CHIP) were tested, using 15 rabbits divided into 3 separate groups of 5 animals each. A normal saline perfusate, containing a standard concentration of 10 mg of methotrexate per liter, was recycled through the peritoneal cavity, transmitting, in every group, 6,000 calories of heat. In model I, heat transmission was achieved by a high temperature gradient, in model II by the increased thermocapacity of a great priming volume of perfusate, using a peritoneal expander, and in model III by a high flow rate. The rectal and oesophageal temperatures were recorded and the indications converted to calories. The bowel showed a statistically higher heat uptake in model III, whereas thermodilution was more important in model I and especially in model II. The results indicated that the ideal model of CHIP must combine efficiency and safety. The temperature gradient must be ample, but within safe limits, only for cost-efficiency reasons. The priming volume ought to be abundant enough to achieve homogeneity and constancy of heating, but not excessive, in order to avoid abdominal distension and bodily thermo-dilution. Under these conditions, the target level of heating, always calculated in calories, must be administered by appropriate adjustment of the perfusion flow rate.

Expression of ets family of genes in systemic lupus erythematosus and Sjogren's syndrome.

We have examined the expression of three members of the ets family of genes, ETS1, ETS2 and ERGB/Fli-1, in lymphocytes from patients with systemic lupus erythematosus and in two murine autoimmune model systems. The ERGB/Fli-1 gene is expressed at a higher level in lymphocytes from autoimmune disease patients than healthy individuals. In addition, we found that the ERGB/Fli-1 gene expression is higher in splenic T-cells from lupus prone mice and in infiltrating lymphocytes in the salivary glands of HTLV-I tax transgenic mice. Taken together, these results suggest that the elevated expression of the ERGB/Fli-1 gene in lymphocytes may be a prelude to the development of autoimmune diseases.

P53 gene mutations in women with breast cancer and a previous history of benign breast disease.

Mutations of the p53 tumour suppressor gene are the most common genetic lesions in human cancers and have been reported in breast cancer as part of the Li-Fraumeni syndrome. In the present study, we determined frequencies and types of the p53 mutations in breast cancer tissues in women with a history of benign breast disease (BBD) identified in Florence, Italy, with (n = 6) or without (n = 10) a family history of breast cancer. Among the cases with a family history of breast cancer and BBD, 2 out of 6 had p53 gene mutations in cancer samples. 1 patient had a mutation at codon 248 and the other had double mutations at codons 243 and 241. In these cases, the p53 gene was also analysed in the tissue samples from previous BBD lesions; however, no mutations were observed (0 out of 6). These results suggest that the p53 mutations occur during advanced stages of tumour progression. In sporadic breast cancer cases with a history of BBD, p53 point mutations were observed of tumour progression. In sporadic breast cancer cases with a history of BBD, p53 point mutations were observed in four samples (4 out of 10). Two of these mutations turned out to be silent changes and one of the samples showed triple mutations at amino acid positions 267, 277 and 296. No p53 gene mutations were found in the breast tumour tissues of 10 additional women from the same area with a family history of breast cancer, but no previous BBD (0 out of 10). Family history of breast cancer does not appear to affect the frequency of p53 mutations in women with a previous history of BBD.

Establishment and characterization of a hepatoblastoma cell-line from a patient with secondary acute myeloid-leukemia.

A girl who had been treated, apparently successfully, with surgery and chemotherapy for a hepatoblastoma, fell ill two years later with what was diagnosed as an AMF M(4). A cell line was established from her peripheral blood. This cell line had epithelial morphology and grew both in suspension culture and as a monolayer. The cells were positive for epithelial surface markers, including the liver-specific alpha-fetoprotein, but not for leukocyte markers. The cell-line's karyotype was markedly abnormal. It did not have any specific aneuploidies or any other aberrations characteristic of leukemias; instead it had gains of 2q and chromosome 20, the most common cytogenetic changes in hepatoblastoma. It is most likely that the patient had a relapse of hepatoblastoma with massive seeding of the blood leading to a leukemia-like picture without, of course, excluding other possibilities.

Regional intraarterial infusion of methotrexate with leucovorin rescue in a rat tumor model.

Walker 2S6 carcinosarcoma was transplanted in the paw of 52 male Wistar rats. A week later, the animals were divided into 6 separate groups and treated in different ways, as follows. Group I (control): no treatment. Group II: femoral artery ligation (FAL). Group III: ligation-intraarterial chemotherapy (LIC) with methotrexate (MTX). Group IV: the same treatment followed by leucovorin rescue (LIC+RES). Group V: tourniquet intraarterial chemotherapy and rescue (TIC+RES). Group VI: the same treatment applied via the iliac artery (iliac TIC+RES). The best results (primary tumor eradication: 8/10 cases, propagation of the tumor to the inguinal region: 4/10 cases, necroses of the distal part of the paw: 0/10 cases, p > 0.05) were observed in group VI, where iliac LIC+RES was performed. Analysis of the treatment's mechanism of action revealed that there is an area of optimal treatment in the middle of the limb, and two peripheral zones of under-treatment. At the proximal zone (inguinal region) propagation of the tumor can occur, whereas at the distal zone necrosis of the paw can be manifested.

Relative paucity of p53 gene-mutations in male breast carcinomas.

Mutations of the p53 suppressor -ene are the most common genetic lesion noted in human cancers and appear to be relatively common (30%) as somatic cell mutations in female breast cancer. p53 mutations have also been frequently reported in familial breast cancers as in Li-Fraumeni syndrome (LFS). Males with breast cancer are far rarer than females. We investigated the mutational spectra of the p53 gene in male breast cancers. Of 10 samples analyzed for p53 mutations in exons 5, 6. 7 and 8, only two showed point mutations corresponding to amino acid residues 248 and 290. One of the point mutations turned out to be a silent change, thus representing only DNA polymorphism. Although the number of male breast cancer samples thus far examined is small, the p53 mutations in male breast cancer (10%), unlike females (30%), does not appear to be as frequent.

Subtraction hybridization cDNA libraries from colon carcinoma and hepatic cancer.

cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 X enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available lambda ZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because lambda libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.

Herpes viruses and duodenal ulcer disease.

Antibodies to herpes simplex virus and Epstein-Barr virus were sought in the sera of 33 patients with endoscopically proved duodenal ulcer and 33 healthy controls. Neither the prevalence nor the antibody levels to both viruses were significantly different between the two groups. These results suggest that herpes viruses may not play a role in the pathogenesis of duodenal ulcer disease.

Combination of hyperthermia and methotrexate in the treatment of transplanted Walker sarcoma.

The purpose of this experimental study was to evaluate the possible synergistic effect of hyperthermia (HPT) and methotrexate (MTX) in Wistar rats transplanted with Walker sarcoma. HPT was induced by microwaves of 432 MHZ frequency transmitted through a source of 2 x 5.5 cm2 surface. The temperature in the tumor was maintained at 43 degrees C which was controlled by an intratumor thermocouple connected with an electronic thermometer. Three experiments were performed. First, in 5 rats HPT only was applied and one rat was used as control. Second, HPT and MTX in combination were given to 6 rats. Third, MTX alone was given to 6 rats. In the first experiment all 5 rats had 50% reduction of their tumors compared to control. In the second experiment complete disappearance of the tumor was noticed and in the third experiment the tumor increased in size. Histologically, in the responding tumors extensive necrosis was seen with thrombosed vessels. We conclude that HPT and MTX are synergistic in treating Walker sarcoma in Wistar rats.

Establishment and characterization of a B-cell line from a patient with acute lymphoblastic leukemia.

A permanent lymphoblastoid cell line was established from the peripheral blood of a child with acute lymphoblastic leukemia. The cell line, designated SDK, grows in a stationary suspension culture, forming aggregates, in RPMI medium supplemented with 10% FCS, with a doubling time of 50-60 h. Immunologic markers and cytological features suggested that the SDK cells should be identified as being of B-cell origin. The cells failed to form rosettes with sheep erythrocytes, did not express T-cell antigens as defined by monoclonal antibodies, and exhibited surface and cytoplasmic immunoglobulin determinants. Chromosome analysis revealed the presence of three cell populations with (a) 46XY; (b) t(8q-; 14q+) or 2p-; 14q+) and (c) cells with unidentifiable markers. SDK demonstrated susceptibility to TPA-induced differentiation toward plasma cells.

Interactions between human fibroblasts and HeLa cells in vitro.

Affinity toward each other was demonstrated in co-cultures between HeLa cells and fibroblasts originating from human tumor stromal or normal tissues. Both cell types in the mixed cultures (ratio 1:1, 1:2, 2:1) proliferated normally as shown by 3H-thymidine labeling index estimation for up to 48 hr of co-culture. At ratios of fibroblasts: HeLa lower than 1:10, fibroblasts were eventually eliminated after serial passaging. It was shown that 3H-nucleotides could be transferred between heterologous cells in either direction. Contact of cells was essential for this phenomenon. Transfer of the label from HeLa to fibroblasts required a longer interaction time and was evidently lower than the transfer from fibroblasts to HeLa. 3H-thymidine incorporated into the DNA of either cell type could not be transferred from one cell to another. The model provides a means for studying neoplastic X normal (or tumour stromal) cell interactions in vitro.

Uncontrolled growth of tumour stromal fibroblasts in vitro.

The disturbance of the growth control mechanisms in tumour cells in vivo may be manifested as uncontrolled growth of the tumour stroma in vitro. Stromal fibroblast-like finite cell lines produced from benign or malignant human breast tissue specimens exhibited cell overlapping which ranged from multilayers to dense piling up colonies, while cells derived from normal tissues exhibited intense contact inhibition of growth and locomotion, under the same culture conditions. 6 out of 13 lines derived from malignant tissues and 2 out of 5 lines derived from benign lesions exhibited the phenomenon of 'periodic appearance of piling up colonies'.