Detection and cloning of potent transforming gene(s) from chewing tobacco-related human oral carcinomas.
High molecular weight DNA isolated from 14 primary tumour tissues of human oral carcinoma patients was analysed for transforming activity by NIH3T3 co-transfection assay using pSV2neo gene as a selectable marker, followed by nude mouse tumorigenicity assay. Ten of the patient tumour tissues demonstrated molecular lesions in myc, ras or/and EGF-R genes, whereas 4 patients did not show tumour associated aberrations in these oncogenes. The G418-resistant transfected cells from 12 of 14 individual patients demonstrated transforming potential by colony formation in soft agar and tumour induction in nude mice within 25-80 days. DNAs from the transfected cells, consequent nude mice tumours and corresponding cell lines, contained human Alu sequences. Southern blot hybridisation with ras, myc, EGF-R oncogenes demonstrated the presence of human H-ras oncogene in one of the 12 sets of nude mice tumours. In contrast, DNA from the other 11 sets of nude mice tumours indicated absence of c-myc, N-myc, L-myc, H-ras, K-ras, N-ras and EGF-R genes on Southern analysis. Further, DNAs from five first cycle tumorigenic transformants were subjected to a second cycle of transfection, and induced tumours in nude mice with a shorter latency period of 21-50 days. The secondary transformants contained discrete human Alu sequences; however, the DNA did not hybridise with myc/ras/EGF-R probes. A genomic library was constructed from a second cycle nude mice tumour, using EMBL-3 as the vector. Four human Alu sequence positive clones were isolated on screening 2 x 10(5) plaques, and one of the recombinant clones subjected to fine restriction mapping using 16 restriction enzymes. The lack of association of the nude mice tumour DNA with myc/ras/EGF-R showing aberrations in the primary human tumour, implies activation of an alternative potent transforming gene(s) in the chewing tobacco-related oral carcinomas in India.
Cell and Developmental Pathology Division, Tata Memorial Centre, Parel, Bombay, India.
D
D
Saranath
L T
LT
Bhoite
M G
MG
Deo
A T
AT
Tandle
J
J
D'Costa
R N
RN
Kolhapure
M K
MK
Govardhan
K
K
Banerjee
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002294:Carcinoma, Squamous Cell / Q000235:genetics*; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D018773:Genes, erbB-1 / Q000235:genetics; D016259:Genes, myc / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009062:Mouth Neoplasms / Q000235:genetics*; D009857:Oncogenes / Q000235:genetics*; D010947:Plants, Toxic; D014030:Tobacco, Smokeless / Q000009:adverse effects*; D014162:Transfection
D004273:DNA, Neoplasm