SVIBOR - Project code: 1-08-017

MINISTRY OF SCIENCE AND TECHNOLOGY

Strossmayerov trg 4, HR - 10000 ZAGREB
tel.: +385 1 459 44 44, fax: +385 1 459 44 69
E-mail: ured@znanost.hr

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Project code: 1-08-017


ACTIVATION AND EXPRESSION OF CELLULAR ONCOGENES AND ONCOGENIC VIRUSES IN HUMAN TUMORS


Main researcher: SORIĆ, JASNA (44124)


Assistants
Type of research: basic
Duration from: 01/01/91. to 12/31/95.
Papers on project (total): 34
Papers on project quoted in Current Contents: 8
Institution name: Središnji institut za tumore i slične bolesti, Zagreb (74)
Department/Institute: Department of molecular genetics, Laboratory for experimental ca
Address: Bijenička 54,
City: 10000 - Zagreb, Croatia
Communication
Phone: 385 (0)1 4561-093,4561-111/1678
Fax: 385 (0)425-647, 425-497
E-mail: jsoric"olimp.irb.hr
Summary: Activation and expression of cellular oncogenes and the integration of oncogenic viruses into host cell genome, play a critical role in the development of the malignant tumors. Increased levels of the c-src oncogene expression have been observed in certain human tumor cell lines and tissues. We have shown that the level of c-src oncogene product, pp60 c-src, kinase activity in T47D cells is increased 10-20 fold over that found in another human breast cancer cellline, MCF7, or chicken embryo fibroblasts, despite similar protein levels in all three cells. Normal human mammary epithelial cells do not express pp60 c-src. Recently, interest has been focused on oncogenes and their role in the responce of malignant cells to cancer therapy. We have shown that tiazofurin (TR) provided synergism with difluorodeoxycytidine (DFDC). DFDC inhibited proliferation in OVCAR-5 human ovarian carcinoma cells and colony formation in PANC-1 human pancreatic carcinoma cells. Further, in HL-60 human leukemia cells DFDC induced differentiation and inhibited proliferation in a dose-dependet manner. TR and DFDC provide synergistic cytotoxycity in rat hepatoma cells and additive in PANC-1 cells. The two drugs together should be helpful in treating leukemias and solid tumors in humans. Multiple fractions of gamma rays (0.5Gy daily, 30 fractions) had previously been found to change the sensitivity of human cervical carcinoma HeLa cells to anticancer drugs. We have shown that preirradiation did not induce either amplification or elevated expression of c-myc or c-Ki-ras oncogenes. Further, there is no correlation between expression of c-myc and c-Ki-ras oncogenes and the acquired resistance to cisplatin. Also, the development of resistance of human larynx carcinoma cells to cisplatin and vincristine does not alter the expression of c-myc or c-Ki-ras oncogenes. We did not find any correlation between expression of these oncogenes and the sensitivity to cisplatin, methotrexate or gamma rays. Therefore, it is not possible to predict the response of the malignant cells to cancer treatment solely on the basis of the presence of activated oncogenes. In addition, we investigate the role of human papillomaviruses (HPV), oncogenic DNA viruses, in benign and malignant neoplasia of the uterine cervix and head and neck tumors. We have found that HPV types 16 and 18 are mainly associated with cervical carcinoma and are frequently detected in an integrated state with the cellular DNA;genome types 6 and 11 were found to be in an episomal DNA form prevailing in benign tumors. C-myc protooncogene was not rearanged or amplified in analyzed cervical lesions. To enable routine screening of premalignant cervical lesions for HPV DNA, we have developed a sensitive method of detecting HPV 6, 16 and 18 in cervical scrapes using dot blot hybridization assay. The method visualised an amount of target DNA as low as 10pg without background. The technique is easy to do, sensitive and specific, and may significantly contribute to the management of cervical dysplasia. We performed the cloning and expression of human microplasminogen (mPlg), a truncated (28kDa) form of plasminogen (Plg). mPlg retained the characteristic properties of the parent protein as redards activation by urokinase (uPA). The two methionine residues of mPlg, M53 and M256, were replaced, respectively, by Q53 and L256, to yield a methionineless protein (mPlgM-) whose catalytic properties were indistinguishable from mPlg itself; the modified mPlgM- was not detectable altered upon exposure to CNBr in formic acid under conditions used for peptide chain cleavage at methionine residues. Replacement of R29 by M29 at the activation site of mPlg yielded a mutant protein resistant to activation by uPA, but able to form an active complex with streptokinase (SK). This mutant was activated by CNBr to a mPlg whose catalytic properties were qualitatively identical to the reference enzyme mPlg.

Keywords: Cellular oncogenes, oncogenic viruses, HPV types 6, 16 ,18, microplasminogen, cloning, expression, integration, differentiation, cytotoxicity, antitumor drugs.

Research goals: Increased levels of c-src oncogenes expression have been observed in certain human tumor cell lines and tissues. T47D human breast cancer cells are genetically unstable with heterogenous chromosome amplification, including chromosome 20, site of the c-src gene. The goal of this research is to determine the tyrosyl kinase activity of pp60 c-src, the oncogene product of c-src gene in T47D, MCF7 human breast cancer cells and normal human mammary epithelial cells. The HPV integration in host cell genome might play an important role in tumorigenic process. There are some evidence that HPV infection might be an etiological factor in the development of human cervical cancer.The presence of specific HPV DNA in biopsies derived from condyloma acuminatum, dysplasias and cervical carcinoma tissues is examined. In addition to this, we are examinating if the integration of the HPV sequences into a host cell genome can activate the cellular oncogenes. We examine the influence of anticancer drugs and irradiation in cell culture on differentiation, cytotoxicity and resistance of human carcinoma cells. Multiple fractions of gamma rays ( 0.5Gy daily, 30 fractions ) had previously been found to change the sensitivity of human cervical carcinoma HeLa cells to anticancer drugs, cisplatin, methotrexate and vincrustine. We examine whether multiple fractions of gamma rays can induce the amplification or over expression of c-myc and c-Ki-ras oncogenes. Further, we examine whether the development of resistance to cisplatin and vincristine changes the expression of c-myc and c-Ki-ras oncogenes and to determine whether there is a correlation between the sensitivity of selected clones to cisplatin, methotrexate and gamma rays.

COOPERATION - PROJECTS



COOPERATION - INSTITUTIONS


  1. Name of institution: Institut "Ruđer Bošković"
    Type of institution: State institute
    Type of cooperation: Joint project
    City: 10000 - Zagreb, Croatia

  2. Name of institution: Farmaceutsko-biokemijski fakultet
    Type of institution: University/Faculty
    Type of cooperation: Joint project
    City: 10000 - Zagreb, Croatia

  3. Name of institution: Prirodoslovno-matematički fakultet
    Type of institution: University/Faculty
    Type of cooperation: Joint project
    City: 10000 - Zagreb, Croatia
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