Biomedical subjects

T G Paulson

Publications and source records attributed to T G Paulson.

7 recordsLinked to original sources

PubMed2001

p16(INK4a) lesions are common, early abnormalities that undergo clonal expansion in Barrett's metaplastic epithelium.

Barrett's esophagus (BE) is the only known precursor to esophageal adenocarcinoma, a cancer of which the incidence has been increasing at an alarming rate in Western countries. p16(INK4a) lesions occur frequently in esophageal adenocarcinomas but their role in neoplastic progression is not well understood. We detected 9p21 loss of heterozygosity, p16 CpG island methylation, and p16 mutations in biopsies from 57%, 61%, and 15%, respectively, of 107 patients with BE. In contrast, no mutations were found in p14(ARF) or p15, and methylation was found in only 4% and 13%, respectively. >85% of Barrett's segments had clones with one (p16+/-) or two (p16-/-) p16 lesions. Both p16+/- and p16-/- clones underwent extensive expansion involving up to 17 cm of esophageal mucosa. The prevalence of established biomarkers in BE, such as 17p (p53) loss of heterozygosity, aneuploidy, and/or increased 4N (tetraploid) populations, increased from 0% to 20% to 44% in patients whose biopsies were p16+/+, p16+/-, and p16-/-, respectively (P < 0.001). Barrett's segment lengths also increased with change in p16 status with a median of 1.5, 6.0, and 8.0 cm for patients with p16+/+, p16+/-, and p16-/- biopsies, respectively (P < 0.001). We conclude that most Barrett's metaplasia contains genetic and/or epigenetic p16 lesions and has the ability to undergo clonal expansion, creating a field in which other abnormalities can arise that can lead to esophageal adenocarcinoma.

Adult↗

PubMed1999

Evolution of neoplastic cell lineages in Barrett oesophagus.

It has been hypothesized that neoplastic progression develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal populations with accumulated genetic errors. Accordingly, human cancers and some premalignant lesions contain multiple genetic abnormalities not present in the normal tissues from which the neoplasms arose. Barrett oesophagus (BE) is a premalignant condition which predisposes to oesophageal adenocarcinoma (EA) that can be biopsied prospectively over time because endoscopic surveillance is recommended for early detection of cancer. In addition, oesophagectomy specimens frequently contain the premalignant epithelium from which the cancer arose. Neoplastic progression in BE is associated with alterations in TP53 (also known as p53) and CDKN2A (also known as p16) and non-random losses of heterozygosity (LOH). Aneuploid or increased 4N populations occur in more than 90-95% of EAs, arise in premalignant epithelium and predict progression. We have previously shown in small numbers of patients that disruption of TP53 and CDKN2A typically occurs before aneuploidy and cancer. Here, we determine the evolutionary relationships of non-random LOH, TP53 and CDKN2A mutations, CDKN2A CpG-island methylation and ploidy during neoplastic progression. Diploid cell progenitors with somatic genetic or epigenetic abnormalities in TP53 and CDKN2A were capable of clonal expansion, spreading to large regions of oesophageal mucosa. The subsequent evolution of neoplastic progeny frequently involved bifurcations and LOH at 5q, 13q and 18q that occurred in no obligate order relative to each other, DNA-content aneuploidy or cancer. Our results indicate that clonal evolution is more complex than predicted by linear models.

Adenocarcinoma↗

PubMed1999

Loss of heterozygosity analysis using whole genome amplification, cell sorting, and fluorescence-based PCR.

Loss of heterozygosity (LOH) is a common genetic lesion found in many human neoplasms. Extending investigation of LOH to large-scale clinical and public health science studies has proven difficult because of the small size and cellular and genetic heterogeneity of human neoplasms, in addition to the challenges associated with increasing throughput. Our approach to LOH analysis was developed using clinical biopsy samples from patients with Barrett's esophagus (BE) and uses flow cytometric cell sorting to increase sample purity, whole genome amplification to increase sample amount, and automated fluorescent genotyping to increase sample throughput. This approach allows LOH assessment at 20 loci in DNA extracted from 1000 flow-purified cells while maintaining accurate and reproducible allele ratios compared with the standard method of using genomic DNA. This method of analysis should allow accurate, reproducible determination of allele ratios in a variety of human tumors and premalignant conditions.

Alleles↗

PubMed1998

Gene amplification in a p53-deficient cell line requires cell cycle progression under conditions that generate DNA breakage.

Amplification of genes involved in signal transduction and cell cycle control occurs in a significant fraction of human cancers. Loss of p53 function has been proposed to enable cells with gene amplification to arise spontaneously during growth in vitro. However, this conclusion derives from studies employing the UMP synthesis inhibitor N-phosphonacetyl-L-aspartate (PALA), which, in addition to selecting for cells containing extra copies of the CAD locus, enables p53-deficient cells to enter S phase and acquire the DNA breaks that initiate the amplification process. Thus, it has not been possible to determine if gene amplification occurs spontaneously or results from the inductive effects of the selective agent. The studies reported here assess whether p53 deficiency leads to spontaneous genetic instability by comparing cell cycle responses and amplification frequencies of the human fibrosarcoma cell line HT1080 when treated with PALA or with methotrexate, an antifolate that, under the conditions used, should not generate DNA breaks. p53-deficient HT1080 cells generated PALA-resistant variants containing amplified CAD genes at a frequency of >10(-5). By contrast, methotrexate selection did not result in resistant cells at a detectable frequency (<10(-9)). However, growth of HT1080 cells under conditions that induced DNA breakage prior to selection generated methotrexate-resistant clones containing amplified dihydrofolate reductase sequences at a high frequency. These data demonstrate that, under standard growth conditions, p53 loss is not sufficient to enable cells to produce the DNA breaks that initiate amplification. We propose that p53-deficient cells must proceed through S phase under conditions that induce DNA breakage for genetic instability to occur.

Aspartic Acid↗

PubMed1997

Maintaining genetic stability through TP53 mediated checkpoint control.

TP53 serves as a key relay for signals elicited by cellular stresses arising from diverse environmental or therapeutic insults. This relay then activates a cell cycle arrest or cell death program, depending on the stimulus and cell type. The absence of TP53 function disables the cell death or arrest programmes, thereby allowing the emergence of variants with various types of genomic alterations. The data discussed focus on two different types of signals that trigger the TP53 relay system. Firstly, TP53 arrests cell cycle progression in response to the types of DNA damage most commonly detected in cells undergoing tumour progression. Secondly, TP53 is activated by specific depletion of ribonucleotide pools, which prevent cells from entering S phase under conditions that could lead to chromosome breakage. The contribution of both responses limits the emergence of genetic variants. The DNA damage induced arrest appears to be triggered by as few as one double strand break in normal human fibroblasts. Analysis of the arrest kinetics after ionizing radiation shows that TP53 activates a prolonged arrest response in cells with irreparable DNA damage and that high efficiency cell elimination is achieved by a process that can be activated over multiple cell cycles. These data indicate that the primary function of the TP53 arrest/apoptosis pathway in response to double strand break is to eliminate damaged cells from the proliferating population, not to allow additional time for lesion repair. However, it remains possible that repair of other types of damage may benefit from TP53 mediated arrest. Analyses in model genetic systems indicate that the absence of TP53 function allows, but does not ensure, a high intrinsic rate of genetic variation and that instability is increased substantially when cells proceed through S phase under inappropriate growth conditions. This implies that inactivation of TP53 function in combination with other genetic alterations, such as oncogene activation, could accelerate genomic instability and tumour progression.

DNA Damage↗

PubMed1996

Microsatellite instability correlates with reduced survival and poor disease prognosis in breast cancer.

Size changes in microsatellite sequences have been detected in many types of cancer, but the influence of this form of genetic instability on disease progression remains unclear. We determined the incidence of microsatellite instability in breast cancer by comparing PCR-amplified sequences from paraffin-embedded samples of normal and tumor tissue from affected individuals. This analysis showed that at least 30% of breast cancers exhibit microsatellite instability (MI). Of importance, MI correlated with indicators commonly associated with poor disease prognosis, including lymph node status, tumor size, and advanced tumor stage. Individuals with MI+ tumors also showed significantly reduced disease-free and overall survival. These data contrast with studies showing that MI correlates with improved prognosis in colon and gastric cancers. We propose that defects resulting in MI promote disease progression and result in a poor prognosis in breast cancer.

Breast Neoplasms↗

PubMed1995

Genetic instability as a consequence of inappropriate entry into and progression through S-phase.

The stability of the mammalian genome depends on the proper function of G1 and G2 cell cycle control mechanisms. Two tumor suppressors, p53 and retinoblastoma (Rb), play key roles in progression from G1 into S-phase. We address the mechanisms by which these proteins mediate a G1 arrest in response to DNA damage and limiting metabolic conditions. Gamma-irradiation induced a prolonged, p53-dependent G1 arrest associated with a long-term increase in the levels of the cdk-inhibitor p21WAFl/Cipl (p21). Microinjection of linear plasmid DNA also caused a G1 arrest. The p53-dependent arrest induced by inhibitors of UMP biosynthesis was reversible and occurred in the absence of detectable DNA damage. Both arrest mechanisms contribute to limiting the formation and propagation of damaged genomes. Cells containing mutant p53 but wild-type Rb do not generate methotrexate (Mtx) resistant variants. However, pre-treatment with DNA damaging agents prior to drug selection resulted in resistant clones containing amplified dihydrofolate reductase (DHFR) genes, suggesting that DNA breakage is a rate limiting step for gene amplification. The Mtx-induced arrest did not occur in cells with non-functional Rb. Rb acts as a negative regulator of the E2F transcription factors, and Rb-deficient primary mouse embryo fibroblasts (MEFs) produced elevated levels of mRNA and protein for key E2F target genes. Failure to prevent entry into S-phase in Rb-/- MEFs exposed to DNA-damaging or nutrient limiting conditions caused apoptosis and correlated with p53 induction. Taken together, these findings indicate a link between p53 and Rb function and suggest that their coordination insures correct entry into S-phase, minimizing the emergence of genetic variants.

Animals↗