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ONCOGENES IN X-RAY INDUCED LYMPHOMAS IN (C3H X C57BL) F1 AND SJL/MICE

Objective

ONCOGENES IN X-RAY INDUCED LYMPHOMAS IN (C3H X C57BL)F1 AND SPONTANEOUS RETICULUM CELL SARCOMA OF SLJ/J MICE.
1. GENERAL OBJECTIVE
IT HAS BEEN SHOWN THAT DNA ISOLATED FROM EITHER NATURALLY OCCURRING HUMAN TUMORS OR FROM HUMAN TUMOR CELL LINES INDUCE MORPHOLOGICAL TRANSFORMATION WHEN TRANSFECTED INTO NORMAL MOUSE NIH/3T3 CELLS. ALMOST ALL THESE TRANSFORMING GENES HAVE BEEN CHARACTERIZED AS MEMBERS OF THE RAS FAMILY OF TRANSFORMING GENES CODING FOR IMMUNO-RELATED PROTEINS DEFINED P21 BY THEIR MOLECULAR WEIGHT. THE GENETIC ALTERATIONS RESPONSIBLE FOR THE ACTIVATION OF THESE ONCOGENES HAVE BEEN LOCATED WITHIN THE ONCOGENE CODING SEQUENCES, EXACTLY WITHIN THE TRIPLET CODING FOR THE 12, 59 OR 61 AMINO ACID OF P21 PROTEINS. THESE RESULTS CLEARLY DEMONSTRATE THAT MUTAGENESIS IS INVOLVED IN THE ACTIVATION OF HUMAN ONCOGENES. AN IMPORTANT QUESTION IS HOW THESE MUTATIONS ARISE. CHEMICALLY- OR PHYSICALLY-INDUCED AS WELL AS SPONTANEOUS ANIMAL TUMORS CAN BE A HELPFUL MODEL FOR STUDYING THEIR ONSET. THE BASIC PURPOSE OF THIS RESEARCH PROPOSAL IS TO ADOPT THESE ANIMAL TUMORS AS MODEL FOR STUDYING THE ROLE OF ONCOGENES IN CANCER ONSET AND DEVELOPMENT.
In a study of radiation carcinogenesis in animals, three different model systems were used to investigate, at the molecular level, the mechanisms of protooncogene activation and their roles in the onset and development of neoplasias and during tumour progression:
X-ray induced lymphomas in (C57 B1/Cne x C3H/Cne) F1 hybrid mice;
spontaneous mouse reticulum cell sarcomas (RCS);
nonmetastatic and highly metastatic Friend leukemia cells (FLC).
2.1. TO ANALYZE THE PRESENCE OF TRANSFORMING GENES IN THYMIC LYMPHOMAS OF F1 HYBRIDS BETWEEN C57BL/CNE AND C3H CNE MICE TREATED WITH X-RAYS, AND IN RETICULUM CELL SARCOMAS IN SJL/J MICE.
2.2. TO MOLECULARLY ISOLATE AND CHARACTERIZE THESE ONCOGENES, IF ANY.
2.3. TO STUDY ONCOGENE ACTIVATION BY CHEMICAL OR PHYSICAL CARCINOGENS IN INBRED STRAINS OF MICE WITH HIGH FREQUENCY OF SPONTANEOUS TUMOURS.
3. PLANS OF OPERATIONS
3.1. 3-MONTHS OLD F1 HYBRIDS BETWEEN C57BL/CNE AND C3H/CNE MICE WILL BE IRRADIATED WITH DOSE OF 6 GY OF X-RAYS (250 KVP, 15 MA, HVL = 1.5 MMCU, 1.35 GY/MIN) ACCORDING TO COVELLI ET AL., RAD. RES. 91: 615, 1982.
3.2. 3-MONTH OLD SJL/J MICE WILL BE IRRADIATED FOLLOWING THE PREVIOUS PROTOCOLS OR TREATED WITH NITROSO-METHYL-UREA OR OTHER CHEMICAL CARCINOGENS.
3.3. DNAS FROM THE CHEMICALLY OR OTHERWISE INDUCED TUMORS, AS WELL AS DNAS FROM SPONTANEOUS RETICULUM CELL SARCOMA FROM SJL/J MICE WILL BE TRANSFECTED INTO NORMAL MOUSE FIBROBLASTS. DNAS ARE EXTRACTED FROM TUMOR TISSUES BY PHENOL-CHLOROFORM PROTOCOLS AND ETHANOL-PRECIPITATED. THE DNA WILL BE RESUSPENDED IN APPROPRIATE BUFFERS AND USED IN TRANSFECTION EXPERIMENTS, WHICH WILL BE CARRIED OUT FOLLOWING PROTOCOLS ALREADY DESCRIBED. APPROXIMATELY 30 UG OF HIGH MOLECULAR WEIGHT DNA IN 1 ML OF 0.25 M CACL2 IS SLOWLY ADDED TO 2 ML OF 250 MH HEPES, PH 7.1,1.5 MH SODIUM PHOSPHATE, WHILE A GENTLE STREAM OF NITROGEN IS BUBBLED THROUGH. AFTER FORMATION OF THE CALCIUM PHOSPHATE PRECIPITATE, THIS DNA SOLUTION IS ADDED TO A 10-CM PETRI DISH IN WHICH 1.5 X 10 TO THE POWER OF 5 NIH/3T3 CELLS HAVE BEEN SEEDED THE DAY BEFORE. AFTER A 22-HR INCUBATION, THE DNA IS REMOVED AND 10 ML OF FRESH DULBECCO'S MODIFIED EAGLE'S MEDIUM CONTAINING 5% CALF SERUM IS ADDED. FOCI ARE COUNTED 2 WEEKS LATER.
3.4. BASED ON THE RESULTS OF ITEMS 3.1 AND 3.2, TRANSFORMED CELLS WILL BE ISOLATED BY CLONING CYLINDER PROCEDURE, CLONED IN AGAR AND CHARACTERIZED FURTHER.
3.5. ANALYSIS OF CLONAL TRANSFORMED CELLS WILL BE PERFORMED BY SOUTHERN BLOT PROCEDURES. TWENTY UG OF HIGH MOLECULAR WEIGHT DNA ARE DIGESTED WITH APPROPRIATE RESTRICTION ENDONUCLEASES AND APPLIED TO HORIZONTAL AGAROSE GELS. SAMPLES ARE ELECTROPHORESED AT 30V FOR 20 HR, BLOTTED TO NITROCELLULOSE SHEETS AND HYBRIDIZED FOR 48 HR TO 2 X 10 CPM OF THE CORRESPONDING NICK-TRANSLATED (P-32)- LABELLED DNA AS DESCRIBED BY SOUTHERN. ONCE THE DONOR SEQUENCES, CARRYING THE TRANSFORMING GENES, WILL BE DEFINED, WE MAY ATTEMPT TO CLONE THEM, FOLLOWING SUITABLE STANDARD PROTOCOLS.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Head of the Unit of Foodborne Zoonoses and Veterinary Epidemiology
Address
Viale Regina Elena 299
00161 Rome
Italy