Experimental Study On The Radiation-induced FHIT Change And The Radiosensitivity Of HepG-2 After FHIT Transfection

Ionizing radiation is a two-edge sword.On the one hand, ionizing radiation is the most common physical carcinogens. Its potential hazard to human's health is increasing with the widely use of nuclear energy. To illuminate the molecular mechanism of radiation carcinogenesis is the key point of oncology, radiation biology and preventive medicine. On the other hand, radiation therapy is one of the most common methods used to treat tumor. Radiation therapy is the use of high-energy radiation from x-rays, y rays, neutrons, and other sources to kill cancer cells and shrink tumors. Radiation may come from a machine outside the body (external-beam radiation therapy), or it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, implant radiation, or brachytherapy).Many genes are involved in the moeculer mechanism of radiation carcinogenesis and radiation therapy. Malignant transformation is a complex multistep process involving numerous genetic changes, which include loss of tumour-suppressor-gene function, oncogene activation, and alteration of modifier genes. A large body of evidence supports double-stranded breaks of nuclear DNA as the most important cellular effect of radiation. This breakage leads to irreversible loss of the reproductive integrity of the cell and eventual cell death. Radiation damage can be directly ionizing;however, in clinical therapy, damage is most commonly indirectly ionizing via free-radical intermediaries formed from the radiolysis of cellular water. Radiation can also affect the processes of the cell cycle necessary for cell growth, cell senescence, and apoptosis (programmed cell death). During the past decade, evidence has accumulated in support that FHIT, as a new tumour suppressor gene, plays a roll in both radiation carcinogenesis and radiation therapy.The FHIT gene is composed of 10 exons and spans approximately 1 Mb ofgenomic DNA. This gene also encompasses the FRA3B fragile region, which is a documented integration site for HPV 16. The FHIT open reading frame (exons 5 through 9) encodes a 16.8-kDa protein that functions as a dinucleoside 5'5'"-Pl, P3-triphosphate (Ap3A) hydrolase enzyme. FHIT inactivation and loss of expression is found in a large fraction of premaligant and malignant lesions.Our previous studies have demonstrated that the FHIT play an important role in y ray-induced BALB/c mice tumor model. And many reports indicated that FHIT may be a potential gene target for tumor gene therapy. To Study the role of fragile histidine triad gene in radiation carcinogenesis and radiation therapy, the experiment is composed of three parts:1. Nowadays, charged microbeam is widely used in the low-dose radiation biology, especially in studying on the bystander effect. To study the changes of a specific gene after radiation, we need a steady method to detect the gene. Single cell reverse transcription ploymerase chain reaction (RT-PCR) is an excellent technology for the detection and quantitation of mRNA within single cells. In this study, we define a successful method to amplify specific mRNA segments from single cells. The modified, using one-step RT-PCR to perform reverse transcription and first round PCR rather than two-step RT-PCR in the classical type,. We show that this new method remains high specificity and is more sensitive than the previous method in amplifying specific genes.2. Many reports showed that ionizing radiation can induce FHIT gene to express truncated transcripts. Dano et al found in radon-induced rat lung tumors, truncated transcripts of FHIT gene is frequently deleted. My institute had developed a model of y rays-induced mice lymphocyte tumors.Using nested RT-PCR, Lin Yahua et al had found that FHIT gene express truncated transcripts one month after irradiated. To investigate the early alteration of FHIT gene irradiated by y-ray, sixty-four BALB/c mice were divided into 0.5,1.0,1.75 Gy irradiation groups and control group at random. Mice were killed 36h and 7d after they were exposed to y-ray. Total RNAs were extracted from blood, thymus glands and bone marrow. The expression of FHIT gene was studied by means of nested RT-PCR. We found that aberrant transcripts of FHITgene were detected in blood, thymus and bone marrow in different irradiation dose groups. The results indicated that irradiation can cause the deletion of fhit gene early after exposed, and may participate in radiation carcinogenesis.3. Radiation therapy combined with gene therapy is a new way to treat tumors. Evidences proved that FHIT took part in the pathway of cell cycle and apoptosis. In 1997, Siprashvili and colleagues found that re-expression of FHIT in FHIT-negative tumour cell lines suppresses the tumorigenicity of these cells in vivo. In this study, cells into which the FHIT gene was introduced and control cells without FHIT were injected into immunosuppressed nude mice. 3 weeks later, the control cells had grown into large tumours but the FHIT-expressing cells showed hardly any tumour development. Subsequently, the effect of re-expression of FHIT was studied in lung-cancer cell lines.Adenoviral transduction of FHIT into these cells caused a significant inhibition of cell growth and increased apoptosis. The increase in exogenous FHIT protein expression corresponded to the decrease in cell proliferation and the increase in apoptotic activity. In addition, these FHIT-expressing cells could no longer form tumours in vivo.Recently, Wang Ya and colleagues reported that an overactivated ATR/CHKl pathway regulated the stronger checkpoint responses shown in Fhit"7" cells, which was responsible for the radioresistance of FhifA cells. These results suggest a model to explain the association between Fhit deficiency and tumor progression. This study used pcDNA3.0 vector to establish pcDNA-FHIT vector that contains the interesting target gene. Using Iipofectamine2000 to transfect pcDNA-FHIT and pcDNA3.0 vectors into human liver cancer cell line HepG-2 and G-418 to get the stable cell lines, we get 2 different cell lines pcDNA-HepG and pcDNA-FHIT-HepG. By studying the cell cycle and apoptosis of different cell lines, we come to the conclusion that combine y ray with FHIT transfection may be an effective way to treat the radioresistant cancer and FHIT may be a potential gene target for tumor treatment.