Suppression of tumorigenicity of breast cancer cells by microcell-mediated chromosome transfer: studies on chromosomes 6 and 11.

Development of breast cancer has been associated with deletions at multiple chromosomal regions, including 6q, 11p, and 11q. In this study we analyzed the effects of the introduction of chromosomes 6 and 11 on the cell phenotype of the breast cancer cell lines MDA-MB-231 and MCF-7. Chromosome 6 induced alterations of in vitro growth properties and suppressed tumorigenicity of MDA-MB-231 cells. Spontaneous reduction of the transferred chromosome allowed mapping of the tumor suppressor gene(s) to region 6q21-q23 and/or 6q26-q27. Clones MCF-7/H6 underwent a senescence process that lasted five months. Chromosome 11 had no effect on MDA-MB-231 cells, although it suppressed tumorigenicity of MCF-7 cells. A MCF-7/H11 clone lacking the short arm of the transferred chromosome retained tumorigenicity, however, tumor cell growth was significantly reduced. These results suggest that each chromosomal arm may contain genes important for the suppression of MCF-7 tumorigenic properties.

Tumor and growth suppression of breast cancer cells by chromosome 17-associated functions.

Losses of functions from chromosome 17 are the most frequent genetic abnormalities in human breast cancer. To assess the biological role of chromosome 17 in the development of breast cancer, we transferred a normal human chromosome 17 to two breast cancer cell lines. No viable clone maintaining an intact chromosome was obtained in either MDA-MB-231 or MCF-7. Only one MDA-231/H17 clone contained the long arm of the transferred chromosome 17. Interestingly, this clone lost the ability to induce tumors in nude mice, indicating that at least one gene mapping to the long arm of chromosome 17 could suppress the tumorigenic phenotype. The p53 protein most likely was responsible for the selective loss of the short arm of the chromosome. Both cell lines have no wild-type p53 activity. MDA-MB-231 carries a single mutant TP53 allele, while MCF-7 carries two wild-type alleles, but p53 protein is excluded from the nucleus. Transfection in both cell lines of vectors expressing wild-type p53 produced only clones with rearrangements of the transfected TP53 complementary DNA. Thus, nonregulated expression of the p53 protein driven by the strong cytomegalovirus promoter may have triggered a rapid process of cell death. Stable expression of a mutant p53 in MCF-7 cells proved that nuclear localization of the protein was possible; however, no progression toward an estrogen-independent tumorigenic phenotype was induced. This work indicates that functional inactivation of the wild-type p53 protein and of the product of a gene located on 17q are essential to the development of breast neoplasms.

Suppression of tumorigenesis by the breast cancer cell line MCF-7 following transfer of a normal human chromosome 11.

Breast cancer development is associated with several genetic abnormalities. Loss of heterozygosity in the short arm of chromosome 11 has been observed in 30% of tumors. We found homozygosity at five chromosome 11 polymorphic loci in genomic DNA of the MCF-7 breast carcinoma cell line, suggesting a possible loss of one chromosome 11. We have studied the transformed and tumorigenic phenotypes of MCF-7 cells following introduction of a normal human chromosome 11 via microcell fusion. MCF-7/H11 cell hybrids, containing chromosome 11, showed in vitro characteristics similar to the parental cell line. However, tumorigenicity in athymic mice was completely suppressed. Since tumor formation by MCF-7 cells is estrogen dependent, we have analysed the expression of the estrogen receptor and of the estrogen-activated gene pS2. No difference was detected between the parental MCF-7 cells and the derived chromosome 11 cell hybrids, indicating that the mechanism of MCF-7 tumor suppression by chromosome 11-associated functions does not directly involve the estrogen/estrogen receptor molecular pathway.

Transfer of human chromosome 3 to an ovarian carcinoma cell line identifies three regions on 3p involved in ovarian cancer.

The molecular pathogenesis of ovarian carcinoma involves altered expression of growth factors, activation of oncogenes and loss of tumor suppressor genes. Loss of heterozygosity on chromosomes 3p, 6q, 11p, 17 and 18q was reported as a significant alteration in ovarian cancer. However, no functional proof has been provided of tumor suppressor activity located in these chromosomal regions. We therefore introduced normal human chromosomes 3 and 11 into an ovarian carcinoma cell line by microcell mediated chromosome transfer. Transfer of chromosome 3 induced senescence and growth arrest as well as suppression of tumorigenicity. Tumors induced by chromosome 3 monochromosomic hybrids consistently lost three small regions on 3p, two of which located in 3p23-24.2 and one located in 3p21.1-21.2, suggesting that these chromosomal regions are important for suppression of tumorigenicity of ovarian carcinoma cells. Transfer of chromosome 11 reduced the in vitro growth properties of ovarian cancer cells but did not significantly affect tumorigenicity. These results provide functional evidence for chromosome 3 tumor suppressor activity in ovarian cancer and define the chromosomal regions on 3p involved in the pathogenesis of this tumor. This experimental system, based on functional effects, may be useful for further delimitation and isolation of critical regions on 3p involved in tumor suppression.

Cloning and characterization of a senescence inducing and class II tumor suppressor gene in ovarian carcinoma at chromosome region 6q27.

Cytogenetic, molecular and functional analysis has shown that chromosome region 6q27 harbors a senescence inducing gene and a tumor suppressor gene involved in several solid and hematologic malignancies. We have cloned at 6q27 and characterized the RNASE6PL gene which belongs to a family of cytoplasmic RNases highly conserved from plants, to man. Analysis of 55 primary ovarian tumors and several ovarian tumor cell lines indicated that the RNASE6PL gene is not mutated in tumor tissues, but its expression is significantly reduced in 30% of primary ovarian tumors and in 75% of ovarian tumor cell lines. The promoter region of the gene was unaffected in tumors cell lines. Transfection of RNASE6PL cDNA into HEY4 and SG10G ovarian tumor cell lines suppressed tumorigenicity in nude mice. When tumors were induced by RNASE6PL-transfected cells, they completely lacked expression of RNASE6PL cDNA. Tumorigenicity was suppressed also in RNASE6PL-transfected pRPcT1/H6cl2T cells, derived from a human/mouse monochromosomic hybrid carrying a human chromosome 6 deleted at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% of the clones of XP12ROSV, a Xeroderma pigmentosum SV40-immortalized cell line, transfected with RNASE6PL cDNA, developed a marked senescence process during in vitro growth. We therefore propose that RNASE6PL may be a candidate for the 6q27 senescence inducing and class II tumor suppressor gene in ovarian cancer.

Promotion of tumour metastases and induction of angiogenesis by native HIV-1 Tat protein from BK virus/tat transgenic mice.

OBJECTIVE: To characterize the T53 cell line and its clones derived from an adenocarcinoma of BK virus (BKV)/tat transgenic mice and to establish the role of native Tat in tumorigenicity, induction of metastases and angiogenesis. DESIGN AND METHODS: Tat was quantified by flow cytometry and chloramphenicol acetyltransferase (CAT) assays. Tumorigenicity and metastatic ability of cell lines were assayed in nude mice. Production of proteases was evaluated by a plasmin chromogenic assay and gelatinase zymography. The angiogenic effect was studied in vivo with conditioned medium from tumour cell lines. RESULTS: Tat protein was detected in tumour cell lines in amounts from 600-7000 molecules/cell. Conditioned medium from tumour cell lines was able to transactivate an LTR-CAT in HL3T1 cells, indicating release of extracellular Tat. Tumour cell lines, inoculated into nude mice induced angiogenic tumours with remarkable recruitment of host endothelial cells. Metastases were detected in lymph nodes, lungs, kidneys, and heart. Cell lines produced relevant amounts of proteases. Conditioned medium implanted in mice with matrigel induced an angiogenic response, enhanced by addition of heparin. Preincubation with an anti-Tat antibody abolished the angiogenic effect. CONCLUSIONS: Tat from cells from BKV/tat transgenic mice promotes tumorigenesis and formation of metastases and induces angiogenic activity. Angiogenesis occurs at physiological concentrations of Tat lower than 20 ng/ml. The effects of Tat on induction of metastases and angiogenesis appear to be mediated by activation of proteases.

Correlation between tumour antigens and malignancy in BKV-transformed hamster cells.

Hamster kidney cells transformed by BK virus (HKBK cells) were studied in order to correlate the membrane tumour antigens to the metastatic capability. The presence of the tumour associated surface antigen (TASA) on the surface of HKBK cells was detected by the immunofluorescence test on live cell suspensions. The metastatic ability was investigated by inoculating HKBK cells subcutaneously (s.c.) into newborn hamsters, s.c., intraperitoneally (i.p.) and in the foot pads into adult hamsters, and s.c. into adult hamsters previously immunized with surface antigens extracted from HKBK cells. The results indicate that there is a correlation between the appearance of tumour antigens on the cell surface and the metastatic ability: HKBK cells at low passage (about 30 subcultures after transformation) showed the capping of TASA in the cell membrane and low metastatic ability, whereas HKBK cells at high passage (about 130 subcultures after transformation) exhibited a diffuse appearance of TASA in the cell surface and were highly metastatic.

Antiangiogenic, antitumoural and antimetastatic effects of two distamycin A derivatives with anti-HIV-1 Tat activity in a Kaposi's sarcoma-like murine model.

The antiangiogenic, antitumoural and antimetastatic effects of two novel sulphonic derivatives of distamycin A, PNU145156E and PNU153429, were studied in a Kaposi's sarcoma-like tumour model obtained by injecting nude mice with cells releasing extracellular HIV-Tat protein, derived from a tumour which developed in a BK virus/tat transgenic mouse. Both PNU145156E and PNU153429 were administered intraperitoneally every fourth day for three weeks at doses of 100 or 50 mg/kg of body weight respectively, starting one day after injecting the tumour cells. Both drugs delayed tumour growth in nude mice, preventing neovascularization induced by the Tat protein. PNU153429 also significantly reduced the number and size of spontaneous tumour metastases. Both effects on tumour growth and metastases were augmented by treating simultaneously nude mice with 7.5 mg/kg of body weight of minocycline given per os daily for four weeks starting four days after injecting the tumour cells. Neither acute nor chronic toxic side-effects were observed during the life span of treated nude mice. Due to their antiangiogenic and anti-Tat effects, these drugs are promising for the treatment of Kaposi's sarcoma in AIDS patients.

HIV type 1 extracellular Tat protein stimulates growth and protects cells of BK virus/tat transgenic mice from apoptosis.

Cells from BKV/tat transgenic mice were characterized for their tumorigenic phenotype in nude and syngeneic BDF mice. The results indicate that the BKV/tat recombinant transgene has a weak tumorigenic potential, mostly predisposing to oncogenesis, and that second events are required for the development of tumorigenicity. Tat is endogenously produced and released by tumor cells. It is taken up by recipient cells directly from the culture medium, without need of cell to cell contact. Extracellular Tat stimulates proliferation of cells from BKV/tat transgenic mice and protects them from apoptosis under conditions of serum starvation. Our results are in agreement with a model in which Tat induces its effects on target cells in two different ways. Growth promotion may require interaction of extracellular Tat with surface receptors eliciting a signal for cell proliferation, whereas intranuclear localization of Tat is necessary for transactivation of viral and cellular genes.

Ultrasonographic study of portal venous system in portal hypertension and after portosystemic shunt operations.

One hundred sixty patients with portal hypertension were examined by means of ultrasonography in order to evaluate the sensitivity of this technique in the diagnosis of intrahepatic portal hypertension and in the detection of portal vein thrombosis. Thirty-eight of these patients were selected for a portosystemic shunt and were reexamined after operation to assess the value of ultrasonography as a screening test for the patency of surgical portosystemic shunts. In patients with intrahepatic portal hypertension the main ultrasonographic findings observed were dilatation of the portal trunk of more than 1.3 cm (56.6% of cases), patency and dilatation of the umbilical vein (5.8%), presence of intra-abdominal collateral vessels (11.6%), splenomegaly with dilatation of splenic vein radicles at the hilus (91.3%), and disappearance of normal caliber variations during respiration in splenic or mesenteric veins (78.5% and 88.4%, respectively). The disappearance of normal caliber variations proved a highly specific and sensitive finding. Partial or total occlusion of the portal trunk was observed at ultrasonography in 19 of 21 (90.5%) patients with portal vein thrombosis. Surgical portosystemic shunts were displayed in 28 of 37 patients (75.7%). Ultrasonography seems to be the most important noninvasive tool in the diagnosis of portal hypertension. In patients selected for surgical portosystemic shunts ultrasonography supplies morphologic data regarding liver parenchyma and abdominal vascular anatomy, and it should be performed as a routine screening test for assessment of surgical shunt patency.

Plasma amino acid patterns in experimental acute hepatic failure: comparison between hepatectomy and liver devascularization in pigs.

To clarify the physiopathologic mechanism leading to a marked increase in aromatic amino acids (AAA) in acute hepatic failure (AHF), we compared two experimental models of AHF. Ten pigs were submitted to one-stage hepatic devascularization (group A); in eight other pigs total hepatectomy was performed (group B). The animals were maintained under constant glucose infusion. The mean survival time in group A was 23 +/- 2 hours; after hepatectomy it was 30 +/- 4 hours. Hepatic coma progressively deepened from 8 +/- 3 hours in Group A animals and was delayed until 17 +/- 5 hours in the anhepatic pigs. AAA, methionine, and tryptophan immediately increased markedly in pigs with liver ischemia. In group B animals, AAA showed a slight increase only 18 hours after hepatectomy, whereas there were no significant differences in methionine and tryptophan. The different amino acid patterns in the two groups of animals demonstrate that hepatocyte necrosis is a major source of plasma amino acids after liver devascularization. The slight increase in AAA after total hepatectomy suggests that a release mechanism from muscular mass is involved in the later stages of the experiment. The onset of coma is related to the increase in AAA rather than to alterations in blood ammonia that did not differ in either group of animals.

Immuno-biologic integration for therapeutic approach in ovarian carcinoma.

The "in vitro" cell growth of 14 ovarian carcinomas was evaluated and related with the clinical-pathologic stage (FIGO) and grade of histologic differentiation of disease, with the patients' immunological pattern and with the relapse and survival rates. We identified 4 different patterns of "in vitro" cell growth (P1, P2, P3 and P4). Their correlation with the clinical-pathologic stages of disease as well as with the recurrence and survival rates was strong: 75% of recurrences in pattern P3 and 100% in pattern P4, while tumours with patterns P1 and P2 did not relapse. Similar results were obtained for survival, in fact 2 of the 3 patients who died from disease had cell growth pattern P4, and the other a P3. A significant correlation was found with basal natural killer cell activity of peripheral blood lymphocytes too: in patients with P1 neoplasia the basal NK cell activity was significantly higher (p < 0.05) while it was significantly lower (p < 0.05) in patients with P4 neoplasia in comparison with the others. We conclude that the "in vitro" biological behaviour of ovarian neoplasia can be regarded as prognostic factors even if patients' basal NK activity represents the most significant prognostic element, directly related to the recurrence rate (p = 0.002).

The surgical management of Crohn's disease of the colon.

The authors analyze their experience in the management of 64 cases of Crohn's disease, located in the colon in 47 cases. The clinical and therapeutical aspects are discussed with particular attention to the surgical problems for which even today there is no general consensus of opinion especially when the colon is involved.

Attempts to isolate BK virus from children affected by various diseases.

Attempts to isolate BK virus (BKV) from specimens (urine, faeces, throat and nasal swabs and cerebrospinal fluid) obtained from 150 children affected by various diseases failed. Sera from the same children contained in 60.7% of cases high titres of antibodies to BKV.

Decrease of cellular growth potential in "in vitro" culture of amnions with premature rupture of membranes.

The "in vitro" cellular growth of 8 amniotic membranes from preterm deliveries with premature rupture of membrane (PROM) in absence of risk factors as cervical or vaginal infection (microbiologic negativity), cervical incontinence and other mechanical factors, was compared with cellular growth of 9 amnions from preterm deliveries without PROM. Amniotic membranes were set up in the Eagle basal medium with Earle salts and heat-inactivated fetal bovine serum (10%), gentamicin 50 micrograms/ml and amphotericin B 0.5 micrograms/ml. The results suggested that the growth potential of the cells (epithelial cells and fibroblasts) obtained from amnions with PROM was lower than that of cells obtained from amnions without PROM. We postulated that the premature rupture of membranes in patients without risk factors for PROM, would be conditioned by an intrinsic decrease of cellular growth potential.

Clinical results of lumbar sympathectomy alone or as a complement of direct arterial surgery.

Six hundred and eighty patients underwent conservative surgery for chronic obliterative arterial disease of the lower limbs during a 15 year period ranging from 1960 to 1974. Two hundred and fifty-three lumbar sympathectomy were performed in 240 patients, 339 patients had direct surgery and in 133 cases lumbar sympathectomy was added to direct surgery. From this experience the authors conclude: 1. Lumbar sympathectomy has a low mortality rate (3 deaths or 1.18%) and no important after effects: in particular no sexual problems are reported. 2. Lumbar sympathectomy for intermittent claudication seems to be useful since it apparently improves symptoms and prognosis of the operated leg. 3. Lumbar sympathectomy for rest pains or necrosis can be useful in 1/3 of the patients with proximal arterial lesions and in 2/3 of the patients with only peripheral lesions. 4. Lumbar sympathectomy should always be added to direct arterial surgery above the inguinal ligament since it improves the patency rate of arterial reconstruction without increasing the operative risk.

[Carcinomas in situ of the breast]. / I carcinomi in situ della mammella.

In situ carcinomas of the breast represent a clinical reality of recent decades that oncologists have to face today. Their observation is rendered increasingly frequent thanks to the improvements made in radiological techniques for early diagnosis and mass screening, and better health education. Reference is made to cases of lobular and ductal in situ cancer observed at the Bologna Surgical Clinic. Recommendations are made with respect to the drawing up of a rational treatment plan.

[Therapy of spontaneous pneumothorax. Results of 20 years' experience]. / La terapia del pneumotorace spontaneo. Risultati di 20 anni di esperienza.

256 spontaneous pneumothorax episodes in 165 patients were treated during the period 1956-1978. Continuous aspiration with drainage of the pleural cavity was employed in 156 cases, and parietal pleurectomy for 36 recurrent or undrainable cases. One patient in the latter group underwent bilateral parietal pleurectomy. The soundness of aspirative drainage on the occasion of the first episode is underscored by the low percentage of recurrences. The indications for pleurectomy are discussed. Its effectiveness is apparent from the good functional results it offers, in addition to an absence of relapses.

[Surgical treatment of colo-rectal tumors. Immediate and long-term results in 1749 cases]. / Considerazioni sulla terapia chirurgica nei tumori colo-rettali. Risultati immediati e a distanza su 1748 casi.

An assessment is made of the development of surgical techniques and results obtained throughout a series of 1748 cases of colon and rectum cancer observed over a period of 40 years. Attempts to improve survival have included an extension of lymph duct resection. This approach has proved successful, though a longer period of study is still needed. Lastly, rectal cancer is examined in terms of survival in function of various biological factors associated with the neoplasia.

[Hepato-portal circulatory changes after splenorenal anastomosis]. / Modificazioni circolatorie epato-portali dopo anastomosi spleno-renale.

Hepatoportal circulation changes after the shunt were examined angiographically in 5 patients subjected to splenoportal anastomosis and splenectomy. Arterioportography was employed in all cases. Catheterisation of the anastomosis was carried out in 1 patient and suprahepatic phlebography in another. The anastomosis was found to be patent in all cases. A hepatopetal portal flow was noted solely in the two patients examined six days after surgery. Disappearance of hepatic portal perfusion was observed in one of these patients on the 15th postoperative day. Controls carried out one month, one year, and five years after the operation showed that the mesenteric-portal flow was fully deviated through the shunt towards the vena cava in all patients. Selective hepatic arteriography showed an inverted portal flow in 3 patients. An increase in the calibre of the trunk of the hepatic artery and the entire intrahepatic artery network was always present.