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Biomedical subjects

A J Levine

Publications and source records attributed to A J Levine.

At least 19 recordsLinked to original sources
PubMed2002

The p53MH algorithm and its application in detecting p53-responsive genes.

A computer algorithm, p53MH, was developed, which identifies putative p53 transcription factor DNA-binding sites on a genomewide scale with high power and versatility. With the sequences from the human and mouse genomes, putative p53 DNA-binding elements were identified in a scan of 2,583 human genes and 1,713 mouse orthologs based on the experimental data of el-Deiry et al. [el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W. & Vogelstein, B. (1992) Nat. Genet. 1, 45-49] and Funk et al. [Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E. & Shay, J. W. (1992) Mol. Cell. Biol. 12, 2866-2871] (http://linkage.rockefeller.edu/p53). The p53 DNA-binding motif consists of a 10-bp palindrome and most commonly a second related palindrome linked by a spacer region. By scanning from the 5' to 3' end of each gene with an additional 10-kb nucleotide sequence appended at each end (most regulatory DNA elements characterized in the literature are in these regions), p53MH computes the binding likelihood for each site under a discrete discriminant model and then outputs ordered scores, corresponding site positions, sequences, and related information. About 300 genes receiving scores greater than a theoretical cut-off value were identified as potential p53 targets. Semiquantitative reverse transcription-PCR experiments were performed in 2 cell lines on 16 genes that were previously unknown regarding their functional relationship to p53 and were found to have high scores in either proximal promoter or possible distal enhancer regions. Ten (approximately 63%) of these genes responded to the presence of p53.

Algorithms↗
PubMed2001

Wrch-1, a novel member of the Rho gene family that is regulated by Wnt-1.

We report the isolation and cloning of the Wrch-1 (Wnt-1 responsive Cdc42 homolog) cDNA. Wrch-1 is a novel gene whose mRNA level increases in response to Wnt-1 signaling in Wnt-1 transformed cells, Wnt-1 transgene induced mouse mammary tumors, and Wnt-1 retrovirus infected cells. Wrch-1 encodes a homolog of the Rho family of GTPases. It shares 57% amino acid sequence identity with Cdc42, but possesses a unique N-terminal domain that contains several putative PXXP SH3-binding motifs. Like Cdc42, Wrch-1 can activate PAK-1 and JNK-1, and induce filopodium formation and stress fiber dissolution. Active Wrch-1 stimulates quiescent cells to reenter the cell cycle. Moreover, overexpression of Wrch-1 phenocopies Wnt-1 in morphological transformation of mouse mammary epithelial cells. Taken together, Wrch-1 could mediate the effects of Wnt-1 signaling in the regulation of cell morphology, cytoskeletal organization, and cell proliferation.

Amino Acid Sequence↗
PubMed2001

Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by oligonucleotide arrays.

Using an oligonucleotide array containing sequences complementary to approximately 3200 full-length human cDNAs and 3400 expressed sequence tags (GeneChip, Affymetrix), mRNA expression patterns were probed in 18 colon adenocarcinomas and 4 adenomas. Paired normal tissue was available and analyzed for each of the tumors. Relatively few changes in transcript expression are associated with colon cancer. Nineteen transcripts (0.48% of those detected) had at least 4-10.5-fold higher mRNA expression in carcinoma compared with paired normal samples, whereas 47 transcripts (1.3% of those detected) had at least 4-38-fold or lower expression in the tumor tissue compared with the normal samples. Some of these differences were confirmed by reverse transcription-PCR. Many of these transcripts were already known to be abnormally expressed in neoplastic tissue in general, or colon cancer in particular, and several of these differences were also observed in premalignant adenoma samples. A two-way hierarchical clustering algorithm successfully distinguished adenoma from adenocarcinoma and normal tissue, generating a phylogenetic tree that appropriately represented the clinical relationship between the three tissue types included in the analysis. This supports the concept that genome-wide expression profiling may permit a molecular classification of solid tumors.

Adenocarcinoma↗
PubMed2001

Response function of a sphere in a viscoelastic two-fluid medium.

In order to address basic questions of importance to microrheology, we study the dynamics of a rigid sphere embedded in a model viscoelastic medium consisting of an elastic network permeated by a viscous fluid. We calculate the complete response of a single bead in this medium to an external force, and compare the result to the commonly-accepted, generalized Stokes-Einstein relation (GSER). We find that our response function is well approximated by the GSER only within a particular frequency range determined by the material parameters of both the bead and the network. We then discuss the relevance of this result to recent experiments. Finally we discuss the approximations made in our solution of the response function by comparing our results to the exact solution for the response function of a bead in a viscous (Newtonian) fluid.

Journal Article↗
PubMed2001

p53 activation in response to microtubule disruption is mediated by integrin-Erk signaling.

The p53 tumor suppressor is activated in response to various stresses driving the cells into growth arrest or apoptosis. We have addressed the question of how disintegration of microtubule system induces activation of p53. Depolymerization of microtubules by colcemid in rat and human quiescent fibroblasts resulted in accumulation of transcriptionally active p53 that caused cell-cycle arrest at the G1/S boundary. The p53 activation correlated with prominent activation of Erk1/2 MAP kinases that resulted from colcemid-stimulated development of focal adhesions. Inhibition of focal contacts development by plating of cells onto poly-L-lysine abrogated both Erk1/2 and p53 activations in colcemid-treated cells, while plating of cells onto fibronectin caused transient up-regulation of p53 even in the absence of colcemid. Pre-treatment of cells with the specific MEK1 inhibitor PD098059 also attenuated colcemid-induced p53 activation and G1 cell cycle arrest. Cell types which either failed to develop focal adhesions in response to colcemid treatment (human MCF-7 epithelial cells), or lacked colcemid-induced sustained Erk activation (primary mouse embryo fibroblasts and 12(1) cells) showed virtually no p53 up-regulation in response to disruption of microtubules during G0/G1. Our results indicate that p53 activation is not triggered by disintegration of microtubule system by itself, but rather originates from some of the consequences of such disintegration, in particular, from the development of focal adhesions leading to activation of Erk signaling pathway.

Animals↗
PubMed2001

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Algorithms↗
PubMed2001

Role of T antigen interactions with p53 in tumorigenesis.

SV40 induces neoplastic transformation by disabling several key cellular growth regulatory circuits. Among these are the Rb- and p53-families of tumor suppressors. The multifunctional, virus-encoded large T antigen blocks the function of both Rb and p53. Large T antigen uses multiple mechanisms to block p53 activity, and this action contributes to tumorigenesis, in part, by blocking p53-mediated growth suppression and apoptosis. Since the p53 pathway is inactivated in most human tumors, T antigen/p53 interactions offer a possible mechanism by which SV40 contributes to human cancer.

Animals↗
PubMed2001

Early results of right ventricular-pulmonary artery conduits in patients under 1 year of age.

OBJECTIVES: Management strategies for the repair of many complex heart defects require the implantation of a valved conduit between the right ventricle (RV) and the pulmonary artery (PA), often using aortic or pulmonary homograft valves. Their limited availability, however, has led to the development and use of new conduits. We retrospectively compared our experience with small homografts in patients of less than 1 year of age with the TissueMed bioprosthetic valved conduit. METHODS: From March 1994 to November 1997 29 patients in their first year of life underwent conduit implantation for complex heart defects. These were retrospectively reviewed in order to determine the incidence of death or conduit stenosis. Seventeen patients received homografts and 12 TissueMed conduits. RESULTS: Diagnoses and operative details including conduit size were similar in the two groups and in all cases complete repair of the underlying defect was carried out. Early post-operative mortality was 4/17 (23.5%) in the homograft group and 3/12 (25%) in the TissueMed group. Echo Doppler evaluation within 1 month of operation showed no right ventricular outflow tract (RVOT) obstruction in any of the survivors. In the TissueMed group 8/9 (77%) survivors have gone on to develop significant RVOT obstruction within 12 months of operation. There have been three late deaths in this group all related to severe RVOT obstruction. Two patients died during an attempt at balloon dilatation and one patient died of progressive right heart failure. Five patients had successful replacement of the TissueMed conduit. One child remains well with no evidence of RVOT obstruction. At operation to replace conduit, or at autopsy, the stenoses were related to the deposition of fibrous tissue at the anastomotic suture lines. In the homograft group none of the survivors developed RVOT obstruction during the first 12 months post-operatively. There was one late death (non-cardiac in origin) and one child is awaiting conduit replacement 40 months after initial implantation for obstruction. CONCLUSIONS: The homograft is a satisfactory conduit for re-establishment of RV-PA continuity in infancy. Further work needs to be undertaken in order to elucidate the mechanisms of early graft failure in bioprosthetic conduits if these are to be a suitable alternative for RV outflow reconstruction in infants.

Blood Vessel Prosthesis↗
PubMed2001

Identification of a mouse homolog of the human BTEB2 transcription factor as a beta-catenin-independent Wnt-1-responsive gene.

The Wnt/Wg signaling pathway functions during development to regulate cell fate determination and patterning in various organisms. Two pathways are reported to lie downstream of Wnt signaling in vertebrates. The canonical pathway relies on the activation of target genes through the beta-catenin-Lef/TCF complex, while the noncanonical pathway employs the activation of protein kinase C (PKC) and increases in intracellular calcium to induce target gene expression. cDNA subtractive hybridization between a cell line that overexpresses Wnt-1 (C57MG/Wnt-1) and the parental cell line (C57MG) was performed to identify downstream target genes of Wnt-1 signaling. Among the putative Wnt-1 target genes, we have identified a mouse homolog of the gene encoding human transcription factor basic transcription element binding protein 2 (mBTEB2). The mBTEB2 transcript is found at high levels in mammary tissue taken from a transgenic mouse overexpressing Wnt-1 (both tissue prior to active proliferation and tumor tissue) but is barely detectable in wild-type mouse mammary glands. The regulation of mBTEB2 by Wnt-1 signaling in tissue culture occurs through a beta-catenin-Lef/TCF-independent mechanism, as it is instead partially regulated by PKC. The Wnt-1-induced, PKC-dependent activation of mouse BTEB2 in C57MG cells, as well as the ability of Wnt-1 to stabilize beta-catenin in these cells, is consistent with the hypothesis that both the noncanonical and canonical Wnt pathways are activated concomitantly in the same cell. These results suggest that mBTEB2 is a biologically relevant target of Wnt-1 signaling that is activated through a beta-catenin-independent, PKC-sensitive pathway in response to Wnt-1.

Animals↗
PubMed2001

Serum 25-hydroxyvitamin D, dietary calcium intake, and distal colorectal adenoma risk.

Vitamin D has recently emerged as a potentially protective agent against colorectal neoplasia. We assessed the associations between dietary vitamin D, plasma 25-hydroxyvitamin D [25(OH)D], dietary calcium, and colorectal adenomas in a large screening sigmoidoscopy-based case-control study in Southern California. Because conversion of serum 25(OH)D to serum 1,25-vitamin D is highly regulated by serum calcium, we also assessed modification of the 25(OH)D-adenoma association by calcium intake. Cases were 473 subjects with a primary adenoma, and controls were 507 subjects who had no adenomas at sigmoidoscopy and no history of adenomas. Compared with those in the lowest quartile of intake, those in the highest quartile of dietary vitamin D had an adjusted odds ratio (OR) of 0.83 [95% confidence interval (CI) = 0.49-1.41] and those in the highest quartile of dietary calcium had an OR of 0.82 (95% CI = 0.49-1.25). There was a suggestion that plasma 25(OH)D may be protective in this population (OR for highest vs. lowest quartile = 0.74, 95% CI = 0.51-1.09). A significant protective effect of 25(OH)D was clearly evident only in those with calcium intakes below (OR = 0.40 for highest vs. lowest quartile, 95% CI = 0.22-0.71, p for trend = 0.005) and above (OR = 1.17, 95% CI = 0.69-1.99, p for trend = 0.94) the median calcium intake.

Adenoma↗
PubMed2000

A role for the polyproline domain of p53 in its regulation by Mdm2.

The p53 protein plays a key role in the cellular response to stress by inducing cell growth arrest or apoptosis. The polyproline region of p53 has been shown to be important for its growth suppression activity. p53 protein lacking the polyproline region has impaired apoptotic activity and altered specificity for certain apoptotic target genes. Here we describe the role of this region in the regulation of p53 by its inhibitor Mdm2. p53 lacking the polyproline region was identified to be more susceptible to inhibition by Mdm2. Furthermore, the absence of this region renders p53 more accessible to ubiquitination, nuclear export, and Mdm2-mediated degradation. This increased sensitivity to Mdm2 results from an enhanced affinity of Mdm2 toward p53 lacking the polyproline region. Our results provide a new explanation for the impaired growth suppression activity of p53 lacking this region. The polyproline region is proposed to be important in the modulation of the inhibitory effects of Mdm2 on p53 activities and stability.

Animals↗
PubMed2000

Generation of oscillations by the p53-Mdm2 feedback loop: a theoretical and experimental study.

The intracellular activity of the p53 tumor suppressor protein is regulated through a feedback loop involving its transcriptional target, mdm2. We present a simple mathematical model suggesting that, under certain circumstances, oscillations in p53 and Mdm2 protein levels can emerge in response to a stress signal. A delay in p53-dependent induction of Mdm2 is predicted to be required, albeit not sufficient, for this oscillatory behavior. In line with the predictions of the model, oscillations of both p53 and Mdm2 indeed occur on exposure of various cell types to ionizing radiation. Such oscillations may allow cells to repair their DNA without risking the irreversible consequences of continuous excessive p53 activation.

3T3 Cells↗
PubMed2000

Physical and functional interaction between p53 mutants and different isoforms of p73.

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro and in vivo with p73 alpha, beta, gamma, and delta. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

Breast Neoplasms↗
PubMed2000

A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13.

Inbred 129 strain mice are predisposed to developing male germ cell tumors (GCTs) of the testes. The inherent genetic defects that underlie male GCT susceptibility in the 129 mouse strain are unknown. GCT incidence is increased in 129 strain males that lack functional p53 protein, and we have used this finding to facilitate the generation of panels of GCT-bearing intercross and backcross mice for genetic mapping analysis. A 129 strain locus, designated pgct1, that segregates with the male GCT phenotype has been identified on chromosome 13 near D13Mit188. This region of murine chromosome 13 may be syntenic to a portion of human chromosome 5q that is implicated in male GCT susceptibility in humans.

Animals↗