Screening of the entire coding region of p53 in low grade lymphoproliferative disorders

Bromidge, Teresa; Howe, Denise

doi:10.1136/mp.53.4.216

Cited by 4 publications

(4 citation statements)

References 5 publications

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“…Transcript‐level analyses of p53, p21, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were performed using semiquantitative RT‐PCR in Cohort II samples, as reported previously . Two different primer sets – p53RS/p53RA and p53FLRS/p53FLR – were used for p53. Glyceraldehyde‐3‐phosphate dehydrogenase served as the PCR internal control and cDNA from the A431 epidermoid cell line served as the positive control.…”

Section: Methodsmentioning

confidence: 99%

Accumulation of inactive p53 protein in oral squamous cell carcinoma: stabilization by protein interaction

Francis

Kumar

Nalinakumari

et al. 2012

European J Oral Sciences

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Our previous study showed that p53 protein is accumulated in >60% of cases of oral squamous cell carcinoma (OSCC) and its overexpression is related to poor prognosis. However, the mechanism behind this is still elusive. The present study attempts to dissect p53 alterations at various levels from gene to function in tumor biopsies to understand whether molecular alterations have any relationship with the accumulation of p53 protein in OSCC. Protein-stabilizing mutations were observed in only 13.8% of the samples. Neither p53 polymorphisms nor human papillomavirus (HPV) infection (<2%) has any major role in augmented expression of p53 protein. Analysis of the p53 transcript demonstrated that alteration in the level of p53 mRNA (10%) is not the major mechanistic cause for accumulation of p53 protein, and p21 transcript status showed that the overexpressed p53 protein is functionally inactive. Immunoprecipitation studies demonstrated that the known interactors of p53, such as MDM2 and HSP70, have no significant role in stabilizing p53 and the significant presence of some unknown interactors, sequestering p53, and leading to the aberrant accumulation of p53. Our study suggests that overexpression of inactive p53 protein could be manifested as a result of sequestering from degradation by an unknown interacting protein rather than by gene or transcript level of alteration.

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Section: Methodsmentioning

confidence: 99%

Accumulation of inactive p53 protein in oral squamous cell carcinoma: stabilization by protein interaction

Francis

Kumar

Nalinakumari

et al. 2012

European J Oral Sciences

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“…This study addressed the extent to which sensitivity for detecting mutations in mixed samples is affected by using the streamlined format (in which the reciprocal mismatches are tested in a single reaction), compared to the T7 promoter on alternate primers format that permits the reciprocal mismatches to be tested in separate reactions. Mutations comprising less than 50% of the sample were not detected using the streamlined format, whereas p53 mutations present at a level of only 20% were easily detected using the format in which the reciprocal mismatches were tested in separate reactions [Bromidge and Howe, 2000].…”

Section: Comparison Of Nirca and Earlier Rnase Cleavage-based Assaysmentioning

confidence: 99%

“…(In contrast to natural or synthetic single-stranded RNA, synthetic doublestranded RNA is remarkably stable, and can be stored frozen for periods of over one year.) This T7 promoter on alternate primers strategy was used to screen the complete p53 coding region as a single target of ~1.2 kb in leukemic blood samples amplified in single-round PCRs [Bromidge and Howe, 2000]. This study addressed the extent to which sensitivity for detecting mutations in mixed samples is affected by using the streamlined format (in which the reciprocal mismatches are tested in a single reaction), compared to the T7 promoter on alternate primers format that permits the reciprocal mismatches to be tested in separate reactions.…”

Section: Comparison Of Nirca and Earlier Rnase Cleavage-based Assaysmentioning

confidence: 99%

RNase cleavage-based methods for mutation/SNP detection, past and present

Goldrick¹

2001

Hum. Mutat.

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Mutation detection based on ribonuclease cleavage of basepair mismatches in single-stranded RNA probes hybridized to DNA targets was first described over 15 years ago. The original methods relied on RNase A for mismatch cleavage; however, this enzyme fails to cleave many mismatches and has other drawbacks. More recently, a new method for RNase-cleavage-based mutation scanning has been developed, which takes advantage of the ability of RNase 1 and RNase T1 to cleave mismatches in duplex RNA targets, when these enzymes are used in conjunction with nucleic acid intercalating dyes. The method, called NIRCA, is relatively low-cost in terms of materials and equipment required. It is being used to detect mutations and SNPs in a wide variety of genes involved in human genetic disease and cancer, as well as in disease-related viral and bacterial genes. This review describes historical and recently developed RNase cleavage-based methods for mutation/SNP scanning.

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“…2 Briefly single stranded RNA is synthesized from opposing strands of patient PCR product and wild type control product, of the entire p53 coding region (from viral promoter sequences incorporated during PCR) and opposing RNA strands are hybridized. Subsequent digestion with RNase and analysis by agarose gel electrophoresis and ethidium bromide staining results in the observation of digested product for those samples containing a mismatch between patient and wild type sequence.…”

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confidence: 99%