A new human p53 homologue.
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Amino Acid Sequence↗
Biomedical subjects
D Sidransky
Publications and source records attributed to D Sidransky.
Microsatellite deoxyribonucleic acid analysis to detect bladder cancer in bladder exstrophy.
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Adult↗
Role of the p16 tumor suppressor gene in cancer.
Since its discovery as a CDKI (cyclin-dependent kinase inhibitor) in 1993, the tumor suppressor p16 (INK4A/MTS-1/CDKN2A) has gained widespread importance in cancer. The frequent mutations and deletions of p16 in human cancer cell lines first suggested an important role for p16 in carcinogenesis. This genetic evidence for a causal role was significantly strengthened by the observation that p16 was frequently inactivated in familial melanoma kindreds. Since then, a high frequency of p16 gene alterations were observed in many primary tumors. In human neoplasms, p16 is silenced in at least three ways: homozygous deletion, methylation of the promoter, and point mutation. The first two mechanisms comprise the majority of inactivation events in most primary tumors. Additionally, the loss of p16 may be an early event in cancer progression, because deletion of at least one copy is quite high in some premalignant lesions. p16 is a major target in carcinogenesis, rivaled in frequency only by the p53 tumor-suppressor gene. Its mechanism of action as a CDKI has been elegantly elucidated and involves binding to and inactivating the cyclin D-cyclin-dependent kinase 4 (or 6) complex, and thus renders the retinoblastoma protein inactive. This effect blocks the transcription of important cell-cycle regulatory proteins and results in cell-cycle arrest. Although p16 may be involved in cell senescence, the physiologic role of p16 is still unclear. Future work will focus on studies of the upstream events that lead to p16 expression and its mechanism of regulation, and perhaps lead to better therapeutic strategies that can improve the clinical course of many lethal cancers.
Animals↗
Degenerate oligonucleotide-primed PCR (DOP-PCR): evaluation of its reliability for screening of genetic alterations in neoplasia.
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DNA Mutational Analysis↗
A comparison between microsatellite and quantitative PCR analyses to detect frequent p16 copy number changes in primary bladder tumors.
We tested 70 primary bladder tumors for altered copy number of p16 (D9S1752) by microsatellite analysis and by a quantitative PCR (QPCR) assay. These two approaches were fully concordant for 53 tumors, including all 39 tumors in which microsatellite analysis detected loss. In addition, the QPCR method detected useful anomalies in 17 additional cases, including those in which D9S1752 was uninformative. QPCR was abnormal in 56 of 70 (80%) cases, whereas microsatellite analysis was abnormal in 39 of 70 (56%) cases. Although QPCR uses more DNA than microsatellite analysis, it represents a rapid, informative technique that can readily detect both chromosome 9p21 deletions and amplifications in primary bladder tumors without the need for electrophoretic separation.
DNA, Neoplasm↗
p16INK4A adenovirus-mediated gene therapy for human head and neck squamous cell cancer.
Inactivation of the tumor suppressor gene p16INK4A is the most common genetic alteration in human head and neck squamous cell cancer (HNSCC), making it an ideal target for gene replacement. We constructed a replication-defective, recombinant adenovirus capable of directing a high level of p16INK4A protein expression (Ad5-p16) to investigate its benefit in treating HNSCC. Initial in vitro experiments in four human HNSCC cell lines demonstrated that Ad5-p16 treatment significantly inhibits cell growth with up to 96% efficiency. Flow cytometric analysis showed that Ad5-p16 induced a maximum G1-S cell cycle arrest of 90%. Subsequent studies in a nude mouse model demonstrated that Ad5-p16 treatment significantly reduced (cell line 011) or stabilized (cell line 012) established tumors when compared with control treatments (P < 0.008). These results demonstrate for the first time a significant antitumor effect of Ad5-p16 against human HNSCC in vivo and support the potential application of Ad5-p16 to treat locally advanced, unresectable, or metastatic head and neck cancer, as well as microscopic residual disease after surgical resection.
Adenoviridae↗
Detailed deletion mapping at chromosome 9p21 in non-small cell lung cancer by microsatellite analysis and fluorescence in situ hybridization.
We examined 82 cases of primary non-small cell lung cancer (NSCLC) for loss of heterozygosity (LOH) at the chromosome 9p21-24 region using 16 microsatellite markers. A total of 52 tumors (63%) displayed LOH, and 25 of these cases displayed LOH for all markers. Two cases had small hemizygous losses confined to the p16 gene and more distal markers, whereas 3 cases had loss proximal to p16 and extended through marker D9S126. This latter region has recently been described as another minimal region of loss at 9p21 in lung cancer. However, homozygous deletion of the p16 gene was observed in 18 of 85 cases, with only 5 cases having large deletions extended into the D9S126 region. Furthermore, we did not observe homozygous deletion at the 9p21 region that excluded the p16 gene. Fluorescence in situ hybridization (FISH) analysis using genomic probes spanning either the p16 or Hel-N1 (located at D9S126) gene was performed in 14 tumors. The results from FISH correlated with the chromosomal mapping data, suggesting that the p16 region is the major target of deletion at chromosome 9p21 in primary NSCLC.
Carcinoma, Non-Small-Cell Lung↗
Microsatellite analysis and telomerase activity in archived tissue and urine samples of bladder cancer patients.
We performed microsatellite analysis and tested telomerase activity in paired tissue and urine of bladder cancer patients from frozen archived samples. DNA obtained from microdissected tumor and urine sediment was analyzed and compared to peripheral lymphocytes for microsatellite alterations (loss of heterozygosity [LOH] or instability) using a panel of 20 microsatellite markers in 15 patients with transitional or squamous cell carcinoma of the urinary tract. Additionally, telomerase activity was determined in 12 microdissected tumor specimens and corresponding frozen urine pellets. Tumor cell DNA was detected by microsatellite analysis (LOH or shift) in at least one marker in 14/15 microdissected tumor specimens and in 13/15 DNA samples obtained from urine sediments. Telomerase activity was present in 11/12 tumor samples but could not be detected in any of the corresponding urine sediments. Frozen archived urine samples are useful for retrospective studies utilizing microsatellite analysis or other PCR-based approaches after DNA extraction. However, the evaluation of telomerase protein activity in stored urine samples appears to be unsuitable.
Carcinoma, Squamous Cell↗
Genomic organization and mutation analysis of Hel-N1 in lung cancers with chromosome 9p21 deletions.
Allelic loss of chromosome 9p21 is common in small cell lung cancer (SCLC), but inactivation of the tumor suppressor gene CDKN2a is rare, implying the existence of another target gene at 9p21. A recent deletion mapping study of chromosome 9p has also identified a site of deletion in non-small cell lung cancer (NSCLC) centered around D9S126. The Hel-N1 (human elav-like neuronal protein 1) gene encodes a neural-specific RNA binding protein that is expressed in SCLC. We have mapped this potentially important gene in lung tumorigenesis to within 100 kb of the D9S126 marker at chromosome band 9p21 by using homozygously deleted tumor cell lines and fluorescence in situ hybridization to normal metaphase spreads. Hel-N1 is, therefore, a candidate target suppressor gene in both SCLC and NSCLC. We have determined the genomic organization and intron/exon boundaries of Hel-N1 and have screened the entire coding region for mutations by sequencing 14 primary SCLCs and cell lines and 21 primary NSCLCs preselected for localized 9p21 deletion or monosomy of chromosome 9. A homozygous deletion including Hel-N1 and CDKN2a was found in a SCLC cell line, and a single-base polymorphism in exon 2 of Hel-N1 was observed in eight tumors. No somatic mutations of Hel-N1 were found in this panel of lung tumors. Hel-N1 does not appear to be a primary inactivation target of 9p21 deletion in lung cancer.
Base Sequence↗
Frequent inactivation of PTEN/MMAC1 in primary prostate cancer.
Sporadic prostate carcinoma is the most common male cancer in the Western world, yet many of the major genetic events involved in the progression of this often fatal cancer remain to be elucidated. Numerous cytogenetic and allelotype studies have reported frequent loss of heterozygosity on chromosomal arm 10q in sporadic prostate cancer. Deletion mapping studies have unambiguously identified a region of chromosome 10q23 to be the minimal area of loss. A new tumor suppressor gene, PTEN/MMAC1, was isolated recently at this region of chromosome 10q23 and found to be inactivated by mutation in three prostate cancer cell lines. We screened 80 prostate tumors by microsatellite analysis and found chromosome 10q23 to be deleted in 23 cases. We then proceeded with sequence analysis of the entire PTEN/MMAC1 coding region and tested for homozygous deletion with new intragenic markers in these 23 cases with 10q23 loss of heterozygosity. The identification of the second mutational event in 10 (43%) tumors establishes PTEN/MMAC1 as a main inactivation target of 10q loss in sporadic prostate cancer.
Chromosomes, Human, Pair 10↗
Nucleic acid-based methods for the detection of cancer.
Continued elucidation of the genetic changes that drive cancer progression is yielding new and potentially powerful nucleic acid-based markers of neoplastic disease. Pilot studies indicate that these markers can be used to detect cancer cells in a variety of clinical settings with unprecedented precision. Nucleic acid-based markers may prove to be valuable tools for early detection of cancer in asymptomatic individuals, for confirmation or exclusion of a cancer diagnosis that is based on suspicious but nondiagnostic clinical material, for assessment of tumor burden in cancer patients, and for assessment of response to preventive approaches applied to healthy individuals who are at high risk of developing cancer. Examples of these markers, their potential applications, and the current practical limitations on their clinical use are reviewed here.
Biomarkers, Tumor↗
Functional analysis of wild-type and malignant glioma derived CDKN2Abeta alleles: evidence for an RB-independent growth suppressive pathway.
The tumor suppressor gene CDKN2A (p16/MTS1/INK4A), which encodes the cyclin-dependent kinase inhibitor p16(INK4a), is a target of 9p21 deletions during the malignant progression of human gliomas. This gene also encodes a second protein product (human p16beta, murine p19ARF), which originates from an unrelated exon of CDKN2A (exon 1beta) spliced onto exon 2 in an alternate reading frame. Cell cycle arrest by p16beta is caused by an as yet unidentified pathway. In order to test the candidacy of p16beta as a glioma suppressor, we replaced p16(INK4a), p15(INK4b) and p16beta wild-type as well as a series of seven glioma-derived p16beta alleles (R87H, A112V, R120H, A121V, G125R, A128A and A128V), into glioma cell lines that had either CDKN2A-/RB+ (U-87MG and U-251MG) or CDKN2A+/RB- (LN-319) endogenous backgrounds and demonstrated that p16beta can act as a functional glioma cell growth suppressor. Moreover, p16beta, but not p16(INK4a) or p15(INK4b) inhibited the growth of RB-negative LN-319 cells, indicating that p16beta likely exerts its effects through an RB-independent pathway. In vitro and in vivo assays of pRB phosphorylation were consistent with this interpretation. Since none of the glioma-derived p16beta mutations inactivated their growth suppressive activities, it appears that mutations in CDKN2A exon 2 (which is shared in the coding sequences of p16(INK4a) and p16beta) likely exclusively target p16(INK4a).
Alleles↗
Somatic mutations of PTEN in glioblastoma multiforme.
Alterations of the PTEN gene occur in glioblastoma multiforme. To determine the frequency of PTEN alteration, 34 consecutive glioblastomas were studied in detail. Sequencing each of the nine exons amplified from tumor DNA revealed 11 mutations. Analysis of polymorphic markers within and surrounding the PTEN gene identified an additional four homozygous deletion mutations. Loss of heterozygosity (LOH) was observed in 25 of 34 (74%) cases. All mutations occurred in the presence of LOH. PTEN was mutated in 44% (15 of 34) of all glioblastomas studied and 60% (15 of 25) of tumors with LOH on 10q. Thus, PTEN appears to be the major target of inactivation on chromosome 10q in glioblastoma multiforme.
Astrocytoma↗
Normal polymorphism in the incomplete trinucleotide repeat of the arginine-rich protein gene.
The arginine-rich protein (ARP) gene was recently cloned and localized to human chromosome band 3p21. Recent reports have suggested that ARP is mutated in a high percentage of different human tumors. We amplified and sequenced the multiple arginine coding area of the ARP gene in primary head and neck, non-small cell lung, and renal cell cancers. We found a high frequency of genetic changes in this region, including a single base pair substitution and deletions of arginine repeats in primary tumors. However, these changes were always present in matched normal controls. Thus, the variations in the ARP trinucleotide repeat region represent normal polymorphisms rather than tumor-specific mutations.
Carcinoma, Non-Small-Cell Lung↗
Identification of a novel region of homozygous deletion on chromosome 9p in squamous cell carcinoma of the lung: the location of a putative tumor suppressor gene.
Cytogenetic and molecular studies have implied the presence of tumor suppressor genes (TSGs) on chromosome 9p that are critical in the development of lung and other cancers. The p16/CDKN2 gene, a cyclin dependent kinase inhibitor, is a well-defined TSG on 9p21. Although the frequency of mutations in the p16/CDKN2 gene has been detected in approximately 30% of non-small cell lung cancer, loss of heterozygosity on 9p has been observed in greater than 70% of non-small cell lung cancers. These and other deletion mapping studies have suggested the existence of additional TSGs on 9p. This study examined chromosome 9p for TSG loci by analyzing 23 squamous cell carcinomas of the lung with 21 microsatellite markers. Loss of heterozygosity was detected in all of the tumors, and homozygous deletions of the p16/ CDKN2 locus were observed in 6 of the 23 tumors (26%). In addition, a novel region of homozygous deletion was detected in six tumors (26%) at D9S126, approximately 2.5 cM proximal to p16/CDKN2. A single tumor contained a homozygous deletion at both the p16/CDKN2 locus and the D9S126 locus. The possibility of homozygous loss was confirmed by multiplex PCR using both the D9S126 marker and a chromosome 9p control marker. Fluorescence in situ hybridization analysis with P1 and cosmid probes containing D9S126 also confirmed these data. The minimum region of homozygous deletion was determined by testing markers immediately proximal and distal to the D9S126 region. The data identify a homozygous loss on the short arm of chromosome 9 suggesting the presence of a novel TSG locus, proximal to p16/CDKN2 and located between D9S265 and D9S259.
Carcinoma, Squamous Cell↗
Microsatellite-based cancer detection using capillary array electrophoresis and energy-transfer fluorescent primers.
The development of sensitive, rapid, and accurate methods and apparatus for high-throughput short tandem repeat (STR) analysis will be critical for the use of microsatellite alteration in cancer screening. Here we show that STR-based bladder cancer diagnosis can be performed using capillary array electrophoresis and two-color labeling with energy-transfer (ET) fluorescent primers. Rapid (< or = 35 min) separations are achieved on capillary arrays using replaceable separation matrices and the allelic ratios are quantitatively determined with a precision of +/- 10%. With this precision, a variation of 20% was considered diagnostically significant. These methods provide a significant improvement in the speed, ease, and precision of STR analyses compared to slab gel electrophoresis.
DNA, Neoplasm↗
Detection of bladder cancer recurrence by microsatellite analysis of urine.
A reliable, noninvasive method for monitoring patients with transitional cell carcinoma (TCC) of the bladder would be of great clinical benefit. Cystoscopy is currently the "gold standard," but it is invasive, expensive and uncomfortable for the patient. Recently, we demonstrated a novel approach for the detection of primary bladder cancer based on microsatellite analysis of urine DNA. To determine the feasibility of this technique for following-up patients with TCC, we tested serial urine samples from 21 patients who had been treated for bladder cancer with 20 polymorphic microsatellite markers in a blinded fashion. We detected recurrent lesions in 10 out of 11 patients and correctly predicted the existence of a neoplastic cell population in the urine of two patients, 4 and 6 months before cystoscopic evidence of the tumor. The assay was negative in 10 of 10 patients who had no evident cancer. Microsatellite analysis of urine sediment represents a novel and potentially powerful clinical tool for the detection of recurrent bladder cancer.
Aged↗
Deletional, mutational, and methylation analyses of CDKN2 (p16/MTS1) in primary and metastatic prostate cancer.
The tumor suppressor gene CDKN2 (p16/MTS1) resides on chromosome 9p21 and encodes a 16 kDa inhibitor of the cyclin-dependent kinases. Inactivation of CDKN2 by homozygous deletion, point mutation, and recently described aberrant methylation in the 5' promoter region may increase progression through the cell cycle in tumors. In this study, we examine the CDKN2 gene for the presence of inactivating alterations in human prostate cancer. Sequence analysis of cell lines revealed no mutation in LNCaP, PC3, and TSU-PR1 and a missense mutation, GAC-->TAC (asp to tyr), in exon 2 of the DU145 cell line at codon 76. No mutations were identified in three primary prostate cancers or in seven lymph node metastases. Loss of heterozygosity (LOH) was analyzed by analysis of microsatellite markers in the vicinity of the CDKN2 gene. LOH was detected in 12 (20%) of 60 primary tumors at one or more loci and in 13 (46%) of 28 metastases. Methylation analysis of the CpG-rich promoter region revealed a dense methylation of CDKN2 in cell lines PC3, PPC1, and TSU-PR1, and this was found to correlate with a lack of mRNA expression by reverse transcription-polymerase chain reaction. A demethylating agent, 5-aza-2'-deoxycytidine, induced reexpression when cells were exposed in vitro. DU145 and LNCaP expressed the CDKN2 transcript and were unmethylated in the promoter region. Three of twenty-four (13%) primary prostate cancers and 1 of 12 metastatic tumors demonstrated promoter methylation. No normal prostate tissues were methylated at the CDKN2 gene promoter. One tumor was found to contain concomitant LOH and promoter methylation indicative of biallelic inactivation. A comprehensive analysis of CDKN2 in prostate cancer reveals that point mutations are infrequent, but gene deletion and methylation combine to inactivate CDKN2 in a subset of tumors. Moreover, alterations in this gene may represent a late event in prostate cancer progression.
Aged↗