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RADIATION CARCINOGENESIS IN VITRO

Objective

THE INDUCTION OF CANCER IS ONE OF THE MAJOR POTENTIAL HAZARDS OF RADIATION TO THE HUMAN POPULATION. MUCH EFFORT HAS BEEN EXPENDED IN THE PAST TO DETERMINE THE QUANTITATIVE RELATIONSHIP BETWEEN RADIATION-DOSE AND INDUCTION FREQUENCY AS A FUNCTION OF L.E.T., DOSE-RATE AND DOSE FRACTIONATION. NEVERTHELESS THE SHAPE OF DOSE-RESPONSE CURVES IN THE LOW DOSE REGION HAS STILL NOT BEEN CLEARLY DETERMINED WITH ALL TYPES OF RADIATION AND SIMPLE ASSUMPTIONS SUCH AS LINEARITY AND DOSE-RATE INDEPENDENCE ARE NO LONGER JUSTIFIED. THE IN VITRO TECHNIQUE HAS PROVED TO REPRESENT A VALUABLE TOOL TO STUDY THE EFFECTS OF LOW DOSES AS WELL AS THE BASIC MECHANISM OF CARCINOGENESIS. INDEED, IN VITRO, IT IS POSSIBLE TO RECORD A SIGNIFICANT NUMBER OF EVENTS IN A REASONABLE TIME. IT IS ALSO POSSIBLE TO OBSERVE THE INDUCTION PROCESS ITSELF IN THE TARGET CELL WITHOUT INTERFERENCE FROM OTHER FACTORS AND TO DETERMINE POSSIBLE RELATIONSHIPS WITH OTHER RADIATION-INDUCED EFFECTS SUCH AS SURVIVAL AND CHROMOSOMAL ABERRATIONS.
THE OBJECTIVE OF THE PROPOSED INVESTIGATION IS TO DETERMINE NEOPLASTIC TRANSFORMATION FREQUENCIES IN CELL CULTURES EXPOSED TO LOW DOSES OF HIGH L.E.T. IONIZING RADIATIONS WHICH PLAY AN IMPORTANT ROLE IN THE RADIATION PROTECTION FIELD SUCH AS ALPHA PARTICLES FROM RADIOACTIVE DECAYS AND NEUTRON BEAMS.
The experimental work mainly centred on determining the dose effect relationship for the induction of oncogenic transformation in cultured mammalian cells exposed to low doses of high linear energy transfer (LET) ionising radiations such as alpha particles from radioactive decay.
In addition, cell survival, growth kinetics and cell density effects after exposure to various type of radiations were also determined. The system used was the C3H10T1/2 mouse embryo fibroblast cell line.
CELLS OF THE STABILIZED C3H10T1/2 CELL LINE, WILL BE USED IN THIS STUDY. THESE CELLS GROW IN MONOLAYER AND DISPLAY HIGH INHIBITION TO CELL CONTACT. TREATMENT WITH CARCINOGENIC AGENTS LEADS TO TRANSFORMATION WITH MACROSCOPICALLY VISIBLE AGGREGATION OF CELLS OF NEOPLASTIC PHENOTYPE "FOCI". THE CELLS WILL BE MAINTAINED ACCORDING TO CONVENTIONAL PROCEDURES USING EAGLES BASAL MEDIUM SUPPLEMENTED WITH 10% FETAL CALF SERUM. VERY STRICT CULTURE AND IRRADIATION PROTOCOL, BY NOW STANDARDIZED AND AGREED BETWEEN THE VARIOUS RESEARCH GROUPS, WILL BE FOLLOWED. THIS INCLUDES CAREFUL CHOICE OF SERUM LOTS, STRICT CONTROL ON CELLULAR DENSITY AND ON NUMBER OF CELLULAR DIVISIONS BEFORE CONFLUENCE AND OTHER PARAMETERS WHOSE CONTROL HAS PROVED TO BE ESSENTIAL IN GETTING A LOW BACKGROUND AND REPRODUCIBILITY OF THE DATA.
SURVIVAL AS WELL AS TRANSFORMATION FREQUENCY WILL BE DETERMINED IN PARALLEL IN EACH EXPERIMENT. SURVIVAL IS ASSESSED AFTER ABOUT 2 WEEKS OF CULTURE AND A CELL IS CONSIDERED TO HAVE SURVIVED WHEN IT GIVES RISE TO A CLONE OF AT LEAST 50 CELLS. THE RATE OF TRANSFORMATION IS EVALUATED AFTER 6 WEEKS. A CELL IS CONSIDERED AS TRANSFORMED WHEN IT HAS PRODUCED A FOCUS OF TYPE II OR III ACCORDING TO THE CRITERIA PROPOSED IN THE LITERATURE (REZNIKOFF ET AL. CANCER RES. 33, 3231, 1979).
WE WILL FIRST STUDY THE EFFECTS OF 5-7 MEV ALPHA PARTICLES FROM THE RADIOACTIVE DECAY. CULTURES IN THE EXPONENTIAL PHASE AND THE PLATEAU PHASE, GROWING ON A THIN MYLAR SURFACE, WILL BE IRRADIATED IN A PURPOSE BUILT CHAMBER. WE WILL FIRST DETERMINE THE DOSE-EFFECT CURVE ON A RANGE OF DOSES FROM 0.1-7. GY AT 0.5 GY/MIN IN ORDER TO OBTAIN A COMPARISON WITH DATA REPORTED IN THE LITERATURE ON OTHER TYPES OF RADIATION. EFFORTS WILL THEN BE CONCENTRATED ON LOW DOSES AND ON VERY LOW DOSE-RATES, (AS LOW AS 10 TO THE POWER OF MINUS 4 GY/MIN). FOR EACH DOSE 30-500 SAMPLES WILL BE USED, DEPENDING ON THE EXPECTED FREQUENCIES. NEUTRON BEAMS FROM ACCELERATOR WILL BE STUDIED AT A SECOND STAGE.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

THE UNIVERSITY OF MILANO
Address
Via Cicognara 7
20133 Milano
Italy