Harini Shandilya

Thermophilic bacterial DNA polymerases with reverse-transcriptase activity

Conserved motifs found in known bacterial polI DNA polymerase sequences were identified, and degenerate PCR primers were designed for PCR amplification of an internal portion of polI genes from all bacterial divisions. We describe here a method that has allowed the rapid identification and isolation of 13 polI genes from a diverse selection of thermophilic bacteria and report on the biochemical characteristics of nine of the purified recombinant enzymes. Several enzymes showed significant reverse-transcriptase activity in the presence of Mg2+, particularly the polymerases from Bacillus caldolyticus EA1, Caldibacillus cellovorans CompA.2, and Clostridium stercorarium.

A method for clone sequence confirmation using a mismatch-specific DNA endonuclease.

Site-directed mutagenesis and polymerase chain reaction (PCR)-based cloning are well-established methods carried out routinely in most modern molecular biology laboratories. Application of these methods requires confirmation of the DNA sequence of the target gene by sequencing of DNA purified from multiple colonies, a laborious process. We have developed an alternative approach to screen DNA amplified directly from colony DNA for both desired and undesired mutations. This approach is based on the use of a plant mismatch DNA endonuclease, Surveyor Nuclease, to directly screen clones derived by site-directed mutagenesis. We have also used this approach to identify error-free clones of three genes from celery cDNA produced by PCR and TOPO® cloning. Sequence confirmation using Surveyor Nuclease provides a fast and simple approach to obtain desired clones from site-directed mutagenesis and PCR-based cloning methods without the necessity of sequencing DNAs purified from multiple clones.

Acquisition of mitochondrial dysregulation and resistance to mitochondrial-mediated apoptosis after genotoxic insult in normal human fibroblasts: a possible model for early stage carcinogenesis

Acquisition of death-resistance is critical in the evolution of neoplasia. Our aim was to model the early stages of carcinogenesis by examining intracellular alterations in cells that have acquired apoptosis-resistance after exposure to a complex genotoxin. We previously generated sub-populations of BJ-hTERT human diploid fibroblasts, which have acquired death-resistance following exposure to hexavalent chromium [Cr(VI)], a broad-spectrum genotoxicant. Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis. Here, we report on the death-sensitivity of subclonal populations derived from clonogenic survivors of BJ-hTERT cells treated with 5 μM Cr(VI) (DR1, DR2), or selected by dilution-based cloning without treatment (CC1). Following Cr(VI) treatment, CC1 cells downregulated expression of the anti-apoptotic protein Bcl-2 and exhibited extensive expression of cleaved caspase 3. In contrast, the DR cells exhibited no cleaved caspase 3 expression and maintained expression of Bcl-2 following recovery from 24 h Cr(VI) exposure. The DR cells also exhibited attenuated mitochondrial-membrane depolarization and mitochondrial retention of cytochrome c and SMAC/DIABLO following Cr(VI) exposure. The DR cells exhibited less basal mtDNA damage, as compared to CC1 cells, which correlates with intrinsic (non-induced) death-resistance. Notably, there was no difference in p53 protein expression before or after treatment among all cell lines. Taken together, our data suggest the presence of more resilient mitochondria in death-resistant cells, and that death-resistance can be acquired in normal human cells early after genotoxin exposure. We postulate that resistance to mitochondrial-mediated cell death and mitochondrial dysregulation may be an initial phenotypic alteration observed in early stage carcinogenesis.

Abstract 3063: Development of a sensitive COLD-PCR method for the detection of EGFR mutations in DNA

COLD-PCR is a method to exponentially amplify mutations in DNA while linearly amplifying wild type sequences. The method can be adapted to multiple mutation detection platforms including Sanger Sequencing and is amenable to development as a diagnostic kit. EGFR EXONS 20 & 21: Conditions for non-biased, selective amplification of DNA with mutations in the tyrosine kinase activation regions of the EGFR gene (exon 19 deletions and the T790M and L858R point mutations) have been developed. Ice COLD-PCR methodology was used for both exon 19 deletions and the exon 21 L858R mutation. Fast COLD-PCR methodology was used for the T790M mutation in exon 20. Using control plasmids containing the T790M and the L858R mutations, COLD-PCR amplification with SURVEYOR® Nuclease/WAVE® HS confirmation, demonstrated a limit of detection (LOD) of 0.05% mutant DNA in wild type DNA. COLD-PCR amplification followed by confirmation of these mutations using Sanger sequencing had a limit of detection of 0.2%. Only 5-10 copies of amplifiable DNA containing the mutation are needed for amplification using COLD-PCR followed by downstream mutation determination. Similar results were obtained using DNA isolated from the cell lines, H1650 and A549, and serum spiked with DNA containing a T790M mutation. EGFR EXON 19: For detection of deletions in EGFR exon 19, another series of plasmids containing 3, 6, 9, 15, and 21 base-pair deletions were constructed. The two 15 base-pair deletions were those which result in the most commonly observed mutation in non-small cell lung cancer, p.E746_A750del15 (c.2235-2249del15 or c.2236-2259del15). For these exon 19 deletions, the SURVEYOR Nuclease/WAVE HS was able to detect the presence of a mutation at 0.05% and Sanger sequencing was able to confirm these deletions at 0.2%. CONCLUSION: These data demonstrate that COLD-PCR enriches low number and low abundance EGFR mutations including point mutations and deletions, and suggests applicability to serum/plasma. COLD-PCR coupled with mutation detection methods such as Sanger sequencing is a new model for EGFR mutation testing with potential applicability for detection of mutations in other genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2011-3063

Abstract 2119: Very high sensitivity detection of K-RAS exon 2 mutations using fast COLD-PCR

Therapeutic agents such as cetuximab and panitumumab are epidermal growth factor receptor (EGFR) antagonists that can be effective in the treatment of colorectal cancer. However some 40% of these tumors have activating K-RAS exon 2 codon 12 and 13 mutations that have a strong association with poor response to EGFR antagonists. Attempts have been made to detect the presence of K-RAS exon 2 mutations in free circulating DNA from plasma/serum from colorectal cancer patients with K-RAS exon 2 mutations in their tumors. The small amount of circulating DNA in serum and the low sensitivity of the detection techniques used have resulted in sporadic success in establishing this correlation. Co-amplification at Lower Denaturation Temperature-PCR (COLD-PCR) is a novel form of PCR that preferentially amplifies mutant alleles in a mutant/wild-type mixture with wild-type alleles in vast excess. Fast COLD-PCR, one of two forms of the method, is applied to point mutations or insertion/deletions that lower the melting temperature (Tm) of the mutant allele. In Fast COLD-PCR, preferential amplification of a mutant allele is achieved by setting the PCR denaturation temperature during each cycle at the critical temperature (Tc) at which mutant DNA duplexes but not wild-type duplexes are denatured. COLD-PCR is normally carried out in a thermocycler with independent temperature control of each well, ensuring that Tc is precisely achieved. Well-to-well temperature variation of standard, bench thermocyclers is often not within the narrow range required to achieve efficient COLD-PCR mutation enrichment. We have developed a variation of the Fast COLD-PCR method that reduces the need for precise well-to-well temperature control. In this study we have taken advantage of the Tm lowering of most of the K-RAS exon 2 mutations in codons 12 and 13 (G>A or G>T), and have successfully applied our variation of Fast COLD-PCR to their enrichment. A plasmid-based model system was established to measure the Limit of Detection (LOD) of the K-RAS exon 2 codon 12 mutation G12S (GGT>GAT). Mixtures of two plasmids, one bearing a wild-type copy of exon 2 and the other the G12S mutation were prepared at ratios of mutant/wild-type ranging from 1:10 to 1:1,000 (0.1%). A total of 10,000 plasmid copies alone or combined with purified human serum DNA were enriched. Two rounds of PCR were performed: standard PCR producing a 218-bp amplicon followed by enrichment using nested Fast COLD-PCR at a Tc of 80.9 °C producing a 161-bp amplicon. SURVEYOR ® Nuclease/WAVE ® HPLC and Sanger DNA sequencing were used to identify the G>A mutation in the enriched PCR product. The LOD for the G12S mutation before enrichment was 1% by SURVEYOR/WAVE and 5% by sequencing. After Fast COLD-PCR enrichment, the LOD was Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2119.

Abstract 3934: Quantitative assessment of mtDNA damage differentiates long-term and short-term survivors with glioblastoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The role of mitochondrial dysfunction in cancer has long been under scientific scrutiny. Emerging evidence suggests that a critical mechanism of anticancer therapy induced apoptosis may be the generation of mitochondrial reactive oxygen species (ROS) that can damage vital biomolecules, including mitochondrial DNA (mtDNA). We have developed methods to quantify mtDNA damage in human cells and undertook this investigation in glioblastoma to determine if there is a correlation between mtDNA damage and patient survival. Fresh-frozen tumor tissue was collected from 17 patients with glioblastoma before initiation of anti-tumor therapy. The patients were classified as short-term survivors (STS, survival 9 months or less) or long-term survivors (LTS, survival 24 months or more). Following initial resection, all of the patients underwent adjuvant therapy, usually consisting of radiation therapy plus temozolomide (1). We modified a protocol to assess mtDNA damage in which quantitative long-range PCR is performed on an 8.9kb segment of the mtDNA circle (2). The amount of amplified DNA generated is inversely proportional to the extent of mtDNA damage that blocks polymerase synthesis. Total DNA was extracted from the samples, quantified and evaluated qualitatively by gel electrophoresis. The amplified mtDNA was normalized to mtDNA copy number, which was determined by Real-Time PCR of the mitochondrial ND1 gene using DNA standards. A statistically significant correlation was found between the level of mtDNA damage and survival. Higher levels of mtDNA damage - less amplifiable mtDNA - were found in the tumors of LTS than in the tumors of STS. The mean amplifiable mtDNA in nanograms per million copies of ND1 was 22.2 for STS tumors and 15.8 for LTS tumors (two-tail t-test P value = 0.038). These results are consistent with the hypothesis that reactive oxygen species must be generated in order to trigger apoptosis in response to chemotherapeutic drugs, or other treatment modalities. LTS glioblastomas have less amplifiable mtDNA, therefore more damage per mtDNA molecule, indicating that these tumors have greater basal ROS-generating metabolism, less effective anti-oxidant buffering, or poor mtDNA repair or elimination mechanisms. In comparison, STS glioblastomas may “shut down” basal mitochondrial production of ROS, possibly by a more pronounced Warburg effect (decrease in oxidative phosphorylation), buffer the ROS via anti-oxidant mechanisms, or more effectively repair or eliminate their damaged mtDNA. Increased amounts of mtDNA damage therefore may indicate susceptibility to anti-cancer regimens in glioblastoma. References: (1) Marko et al., Genomics 91 (2008) 395-406. (2) Santos et al., 2006, Methods in Molecular Biology, DNA Repair protocols: Mammalian Systems, 2nd Edition, 183-199. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3934.

Abstract 122: Mitochondrial dysregulation and cellular death resistance in response to genotoxic stress

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cellular death resistance is at the foundation of neoplastic progression. Our objective is to identify mechanisms by which genotoxin exposure of normal human cells may lead to intrinsic alterations allowing for selection of survivors with a growth-advantaged phenotype. Certain forms of hexavalent chromium [(Cr(VI)] are human respiratory carcinogens/oxidizing agents. We have generated populations of BJ-hTERT fibroblasts (stably transfected with the telomerase gene), which have acquired resistance to Cr(VI)-induced death, that was not related to DNA adduct formation. In the present study, subclones were derived from clonogenic survivors of BJ-hTERT cells treated with either 0 µM (CC) or a single exposure to 5 µM (DR1 and DR2) Cr(VI). We investigated caspase 3 cleavage in these lines after 24 h exposure to Cr(VI). CC cells exhibited a >70 fold increase in cleaved caspase 3 expression after Cr(VI) treatment, accompanied by substantial release of mitochondrial cytochrome c, consistent with induction of the mitochondrial apoptosis pathway. In sharp contrast, DR1 and DR2 cells showed significant resistance to Cr(VI)-induced caspase 3 cleavage, which was ∼10 fold increase as compared to control, and Cr(VI)-induced cytochrome c release was negligible. Apoptotic resistance was not related to differences in either hTERT or p53 protein levels, which were similar among the cell lines before and after Cr(VI) treatment. We next investigated potential intrinsic mitochondrial alterations that may contribute to the observed apoptosis resistant phenotype. We measured mitochondrial (mt) DNA copy number by RT-PCR of the mtND1 gene, and found no difference among the cell lines, before or after Cr(VI) treatment. Notably, mtDNA is highly susceptible to oxidative damage. Amplification of an 8.9 kb mtDNA product has been shown to be inversely proportional to mtDNA damage. Intriguingly, our data show that the CC cells display 10-20% less amplifiable mtDNA, as compared to the DR cells (reaching statistical significance in DR1), with no effect of Cr(VI) treatment. Mitochondrial spare respiratory capacity (SRC) has been associated with maintenance of a cellular energy reserve in the face of oxidative stress. We measured basal SRC in the CC and DR cells after injection of 1.5 µM FCCP, a respiratory chain uncoupler. Of note, DR1 and DR2 cells showed a significant, ∼2-fold increase in oxygen consumption rate (OCR) as compared to CC cells, with no difference in basal OCR. Taken together, these data indicate that DR cells have both decreased mtDNA damage and increased SRC, which may be related to their intrinsic death resistance. In conclusion, our data suggest that cell survival after a single genotoxic insult involves the selection of cells with intrinsic mitochondrial dysregulation, leading to death resistance, which may play a role in neoplastic progression. Supported by NIH grants CA107972 to SC and supplement to KW, ES05304 and ES09961 to SRP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 122.