Inter-alu PCR detects high frequency of genetic alterations in glioma cells exposed to sub-lethal cisplatin

Abstract

Increased genomic instability contributes to higher frequency of secondary drug resistance and neoplastic progression in tumors as well as in cells exposed to sub-lethal concentrations of chemotherapeutic agents. We have used PCR based DNA fingerprinting techniques of randomly amplified polymorphic DNA (RAPD) and inter-alu PCR to study this phenomenon in the tumor genome. The choice of the primer, either random (for RAPD) or specific (inter-alu PCR) can determine the nature of alterations being assessed. We have compared the inter-alu PCR and RAPD profiles of U87MG glioblastoma cells exposed to sequentially increasing low doses of cisplatin for 24 passages to that of untreated controls. Inter-alu PCR, with 2 primers, demonstrated a number of alterations in the treated cells, in the form of loss / gain and changes in the intensity of bands. No changes were observed by RAPD analysis with 5 primers, however, indicating a preferential increase in the alu mediated recombination frequency in the treated cells (p 5 1.866 3 10 24 ). The number of changes observed with respect to the corresponding leucocyte DNA in the inter-alu PCR profile of 26 primary tumors (Grade II 5 13; Grade IV 5 13), resected before chemotherapy, for the 2 inter-alu primers was very small. We present a novel application of the inter-alu PCR in detecting alterations in long term cultured cells at low dose exposure to a chemotherapeutic agent. Our results suggest that alu mediated recombination may be important in cells exposed to sub-lethal doses of cisplatin but not in the genesis of primary glioma. ' 2005 Wiley-Liss, Inc.

Frequent loss of heterozygosity and altered expression of the candidate tumor suppressor gene 'FAT' in human astrocytic tumors

“…While RAPD primers define distinct loci, their selection is random and not locus based. We have also used this technique to measure the extent of intra-tumor genetic heterogeneity [11,12] and genomic instability in high and low grade tumors [13] and the role of repeat sequences in the generation of instability [14,15]. This technique scans the whole genome for complimentarity at any locus and thus enhances the chances of detecting novel altered genomic regions in tumors.…”

AluScan: a method for genome-wide scanning of sequence and structure variations in the human genome

“…Individual Alu-transposons in the human genome are on the average only 15 - 20% divergent from each other, and PCR primers complementary to the Alu consensus sequence have been employed for inter-Alu PCR [14-18]. Likewise PCR primers based on consensus sequences in the AluJ, AluS and AluY subfamilies could also be devised.…”

“…Inter-Alu PCR is a useful method for isolating human DNA in the presence of animal DNA [14], linkage mapping [15], creation of human specific probes and fingerprints [16], and detection of mutator phenotypes [17] or high frequency genetic alterations [18]. The general strategy of the method is to employ a single PCR primer based on the Alu consensus sequence to amplify the sequence between two Alu elements.…”

A Customized Simulated Annealing Suitable for Primer Design in Polymerase Chain Reaction Processes

“…Studies of gene expression level (Isenbarger et al, 2008;Dixon et al, 2007), loss of allelic heterozygosity (LOH) (Vladušić et al, 2010;Chih-Ming et al, 2009;Franko et al, 2008;Saelee et al, 2008), microsatellite instability (MSI) (Eveno et al, 2010;Bertagnolli et al, 2009), microdeletions (Kolb et al, 2010;Pasmant et al, 2009), quantification of small ncRNAs (Ro et al, 2006;Berezikov et al, 2006) and detection of lowlevel mutations (Milbury et al, 2009) are a few examples of the popularity, success and diversity of uses of PCR. Some of the examples can be characterized as quantitative PCR or Real Time PCR (Lan et al, 2009;Roux, 2009;VanGuilder et al, 2008;Pattyn et al, 2003;Vandesompele et al, 2002) or quantitative multiplex PCR (Sasaki et al, 2010;Wang et al, 2009;Castellsagué et al, 2008), Inter-Alu PCR (Bonafè et al, 2001;Srivastava et al, 2005), or COLD-PCR (Milbury et al, 2009) and miniprimer PCR (Isenbarger et al, 2008).…”