Cancer Letters

Cancer Letters

Volume 125, Issues 1–2, 13 March 1998, Pages 31-34
Cancer Letters

Report of a hitherto unreported sequence present and expressed in a cancer cell line

https://doi.org/10.1016/S0304-3835(97)00501-6Get rights and content

Abstract

During the amplification of a 202 base pair (bp) fragment around the 61 st codon of the N-ras gene, an extra band of about 150 bp in length was observed when genomic DNA from a human epidermoid carcinoma cell line was used as template. This fragment was cloned and sequenced. However, this sequence was not found in the databank. RT-PCR experiments indicated that the sequence is expressed in the cells about 8 h after serum induction. The relevant RNA hybridized to one strand of the sequence but not to the other.

Introduction

During polymerase chain reaction (PCR)-based amplification of a 202 bp fragment of the N-ras gene around the 61 st codon, an additional fragment was routinely observed when DNA from a human cancer cell line, Hep2, was used as template. Single cell cultures were generated from the cell line and when DNA prepared from these subcultures was used as template in PCR reactions, the extra band always showed up, though it was absent when DNA extracted from human tissue was used as template. Cancer cell lines harbour many mutations, translocations and amplifications and such changes in this cell line have probably created extra sites of homology for the primers being used. As these changes often take place in genes which are relevant to the process of carcinogenesis, we decided to investigate the amplified region more closely. The sequence of this fragment has not been reported before. RT-PCR experiments indicate that part of this sequence is expressed at about 8 h after serum induction of Hep2 cells.

Section snippets

Cell culture

Human epidermoid carcinoma cell line Hep2 was cultured in DMEM supplemented with 2% fetal calf serum and antibiotics (100 IU/ml penicillin, 100 μg/ml streptomycin and fungizone).

DNA isolation

DNA was isolated by digestion of cells suspended in Tris (10 mM), EDTA (5 mM) and NaCl (150 mM) with proteinase K (50 μg/ml) and SDS (0.5%) at 50°C for 4 h. The digests were extracted with equal volumes of buffer-saturated phenol several times followed by phenol–chloroform and chloroform. DNA was precipitated first with

Results and discussion

Fig. 1 shows the amplification product after PCR of genomic DNA from two different sources using primers A and B. When the control human blood DNA was used as template, the expected band of 202 bp was obtained. However, when the Hep2 DNA was used as template, an extra band (X) of about 150 bp in length was obtained. When these PCR products were hybridized with the [α-35S]dATP-labelled 202 bp fragment, X failed to light up indicating that it was not a truncated version of the 202 bp band (data

Acknowledgements

Financial assistance from ICMR is acknowledged. N.D. is a CSIR senior research fellow.

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