Posts
Allelic loss of 6q25-27, the PARKIN tumor suppressor gene locus, in cervical carcinoma from northern Indian population
Background:
Frequent loss of heterozygosity (LOH) within genetically defined chromosomal regions is considered an indication of the presence of a putative TSG. Several loss of heterozygosity (LOH) studies have indicated that the chromosome 6q25-q27 region is frequently altered in a variety of human cancers.
Recently, Parkin, a gene implicated in autosomal recessive juvenile Parkinsonism, was found to be a target of LOH at chromosome 6q25-q27 in breast, ovarian and lung tumorigenesis. Although various deletions and point mutations have been described in patients with early onset of Parkinsonism, mutation analysis failed to identify somatic point mutations in any of the breast or ovarian tumors with LOH at the Parkin/FRA6E locus examined.However, truncating and homozygous deletions were identified in the lung adenocarcinoma cell lines Calu-3 and H-1573. RT–PCR of Parkin on a panel of cell lines derived from breast, kidney, cervical and prostate cancers revealed that Parkin is also genetically altered in cancer types other than ovarian cancer.
Objective:
With these background information the present study is aimed to define regions of allelic loss at human chromosome 6q25–q27 in cervical cancer patients from northern India.
Materials and Methods:
Tissue samples: One hundred and five cervical tissue biopsies of carcinoma patients and their matched control samples (blood/precancerous lesions) were collected from Maulana Azad Medical College and associated LNJP Hospital, New Delhi and was immediately stored in -80oC.
DNA extraction: DNA was extracted from the above frozen cervical tissue biopsies and their matched control samples by standard phenol-chloroform method, then dissolved and stored in TE buffer. Finally, purity and concentration of extracted DNA were analyzed by gel electrophoresis and ultraviolet spectrophotometry.
Microsatellite Marker selection and PCR amplification: Three microsatellite marker sites: D6S1599, D6S305 and D6S1008 in chromosome 6q25-27 were selected to detect LOH of Parkin gene. D6S1599 and D6S305 are intragenic markers which are present in Parkin introns 2 and 7, respectively where as D6S1008 is present in the flanking region at telomeric end. Primer sequences are available at the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov/). PCR amplification was carried out in a final volume of 25 μL, containing 50 ng DNA, 2.5 μL of 10X PCR Buffer, 1.5 mmol/L MgCl2, 0.5 μmol/L of each primer, 200 μmol/L of each dNTP, and 1 U Taq DNA polymerase. The amplification conditions were as follows: an initial incubation at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, at 58°C , 62°C and 57°C for 1 min for above mentioned primers respectively, at 72°C for 1 min, and a final extension at 72°C for 5 min.
LOH analysis: The amplified PCR products were denatured at 95°C for 5 min and run on 8% denaturation polyacrylamide gel (1xTBE buffer, circulatory water) at a voltage of 500 V, and power of 45 W for 3.5 h. Silver staining was performed as previously described in Laboratory manual by Sambrook.
Determination of Parkin LOH: The heterozygous genomic allele was targeted for LOH information analysis. LOH was defined as a complete loss or up to 40% decreased relative density of silver staining bands of PCR products in cervical cancer samples compared to their matched control samples (blood/precancerous lesions.
Statistical Analysis: LOH found in two intragenic markers (D6S1599, D6S305) was compared with the marker of flanking region (D6S1008) using the Chi-Square test. P<0.05>Parkin gene in 3 microsatellite sites are shown in Table 1. 59 of 105 (56%) samples showed LOH in at least one locus in the region examined. The percentage of LOH for each of the three markers ranged from 25% (D6S1008) to 48% (D6S305). The most frequent site was D6S305, accounting for 48%. Presence of LOH was significantly higher in both the intragenic markers (D6S1599 and D6S305) as compared with the locus of flanking region (D6S1008) with their p value 0.04 and 0.003, respectively.
Background:
Frequent loss of heterozygosity (LOH) within genetically defined chromosomal regions is considered an indication of the presence of a putative TSG. Several loss of heterozygosity (LOH) studies have indicated that the chromosome 6q25-q27 region is frequently altered in a variety of human cancers.
Recently, Parkin, a gene implicated in autosomal recessive juvenile Parkinsonism, was found to be a target of LOH at chromosome 6q25-q27 in breast, ovarian and lung tumorigenesis. Although various deletions and point mutations have been described in patients with early onset of Parkinsonism, mutation analysis failed to identify somatic point mutations in any of the breast or ovarian tumors with LOH at the Parkin/FRA6E locus examined.However, truncating and homozygous deletions were identified in the lung adenocarcinoma cell lines Calu-3 and H-1573. RT–PCR of Parkin on a panel of cell lines derived from breast, kidney, cervical and prostate cancers revealed that Parkin is also genetically altered in cancer types other than ovarian cancer.
Objective:
With these background information the present study is aimed to define regions of allelic loss at human chromosome 6q25–q27 in cervical cancer patients from northern India.
Materials and Methods:
Tissue samples: One hundred and five cervical tissue biopsies of carcinoma patients and their matched control samples (blood/precancerous lesions) were collected from Maulana Azad Medical College and associated LNJP Hospital, New Delhi and was immediately stored in -80oC.
DNA extraction: DNA was extracted from the above frozen cervical tissue biopsies and their matched control samples by standard phenol-chloroform method, then dissolved and stored in TE buffer. Finally, purity and concentration of extracted DNA were analyzed by gel electrophoresis and ultraviolet spectrophotometry.
Microsatellite Marker selection and PCR amplification: Three microsatellite marker sites: D6S1599, D6S305 and D6S1008 in chromosome 6q25-27 were selected to detect LOH of Parkin gene. D6S1599 and D6S305 are intragenic markers which are present in Parkin introns 2 and 7, respectively where as D6S1008 is present in the flanking region at telomeric end. Primer sequences are available at the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov/). PCR amplification was carried out in a final volume of 25 μL, containing 50 ng DNA, 2.5 μL of 10X PCR Buffer, 1.5 mmol/L MgCl2, 0.5 μmol/L of each primer, 200 μmol/L of each dNTP, and 1 U Taq DNA polymerase. The amplification conditions were as follows: an initial incubation at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, at 58°C , 62°C and 57°C for 1 min for above mentioned primers respectively, at 72°C for 1 min, and a final extension at 72°C for 5 min.
LOH analysis: The amplified PCR products were denatured at 95°C for 5 min and run on 8% denaturation polyacrylamide gel (1xTBE buffer, circulatory water) at a voltage of 500 V, and power of 45 W for 3.5 h. Silver staining was performed as previously described in Laboratory manual by Sambrook.
Determination of Parkin LOH: The heterozygous genomic allele was targeted for LOH information analysis. LOH was defined as a complete loss or up to 40% decreased relative density of silver staining bands of PCR products in cervical cancer samples compared to their matched control samples (blood/precancerous lesions.
Statistical Analysis: LOH found in two intragenic markers (D6S1599, D6S305) was compared with the marker of flanking region (D6S1008) using the Chi-Square test. P<0.05>Parkin gene in 3 microsatellite sites are shown in Table 1. 59 of 105 (56%) samples showed LOH in at least one locus in the region examined. The percentage of LOH for each of the three markers ranged from 25% (D6S1008) to 48% (D6S305). The most frequent site was D6S305, accounting for 48%. Presence of LOH was significantly higher in both the intragenic markers (D6S1599 and D6S305) as compared with the locus of flanking region (D6S1008) with their p value 0.04 and 0.003, respectively.
Results:
59 of 105 (56%) samples showed LOH in at least one locus in the region examined. The percentage of LOH for each of the three markers ranged from 25% (D6S1008) to 48% (D6S305). The most frequent site was D6S305, accounting for 48%. Presence of LOH was significantly higher in both the intragenic markers (D6S1599 and D6S305) as compared with the locus of flanking region (D6S1008) with their P value 0.04 and 0.003, respectively.
Conclusion:
Chromosomal region 6q25–q27 has frequently undergone deletions in a wide spectrum of human neoplasms, such as melanoma, ovarian cancer, breast cancer, non-Hodgkin’s B cell lymphoma, and several other types of cancer.
Our findings suggest that Parkin is a strong candidate TSG located at human chromosome 6q25–q27 and that its reduced expression and inactivation may play a major role in the progression of cervix carcinoma.
No comments:
Post a Comment