Ionizing radiation exposure results in up-regulation of Ku70 via a p53/ataxia-telangiectasia-mutated protein-dependent mechanism.

Genome damaging events, such as gamma-irradiation exposure, result in the induction of pathways that activate DNA repair mechanisms, halt cell cycle progression, and/or trigger apoptosis. We have investigated the effects of gamma-irradiation on cellular levels of the Ku autoantigens. Ku70 and Ku80 have been shown to form a heterodimeric complex that can bind tightly to free DNA ends and activate the protein kinase DNA-PKcs. We have found that irradiation results in an up-regulation of cellular levels of Ku70, but not Ku80, and that this enhanced level of Ku70 accumulates within the nucleus. Further, we uncovered that the postirradiation up-regulation of Ku70 utilizes a mechanism that is dependent on both p53 and damage response protein kinase ATM (ataxia-telangiectasia-mutated); however, the activation of DNA-PK does not require Ku70 up-regulation. These findings suggest that Ku70 up-regulation provides the cell with a means of assuring either proper DNA repair or an appropriate response to DNA damage independent of DNA-PKcs activation.

Detection of K-ras mutations in resected primary leiomyosarcoma.

Mutation of the K-ras oncogene occurs frequently in human malignancy. However, there are few reports concerning K-ras mutations in soft-tissue sarcoma, including leiomyosarcoma. We therefore designed a study to determine the prevalence of mutations in the first exon of K-ras in leiomyosarcoma and to evaluate its prognostic potential. Fifty-one leiomyosarcomas were reviewed, and their diagnoses were confirmed on pathological review. Tissue blocks were retrieved, and new sections were prepared for confirmation of diagnosis. Additional tissue sections were used for DNA isolation. PCR and denaturing gradient gel electrophoresis (DGGE) were used to detect K-ras mutations in the first exon of genomic DNA isolated from the specimens. Seven (14%) K-ras mutations were detected using DGGE. Subsequent sequencing of the K-ras gene from each of the mutated tumors confirmed the DGGE results in each case. The median survival for patients whose tumors did not contain mutations of K-ras was 42 months (n = 42) versus 25 months (n = 7) for those with mutations (P = 0.06). However, patients with stages I and II tumors had a median survival of 82 months (n = 28) compared to 28 months for those with stages III and IV disease (n = 20, P = 0.02). The results suggest that K-ras codon 12 mutations are uncommon in leiomyosarcoma; however, when such mutations are found, there is a trend toward worse survival. Furthermore, the data confirm that stage is a significant prognostic indicator.

Distance constraints and stereospecific alignment requirements characteristic of p53 DNA-binding consensus sequence homologies.

We present evidence in favor of the position that some mutant p53 proteins retain the ability to trans-activate downstream genes through p53 DNA-binding consensus sequence (CS) homologies. We tested one cell line possessing high levels of mutant p53 and found that this mutant p53 is highly active in trans-activating one CS homology, moderately active in trans-activating a second sequence and inactive in modulating a third sequence. We tested a second cell line, also possessing high levels of mutant p53 and found the same pattern of activation. In addition we find that inter-motif distance [represented by N in RRRCWWGYYY(N)RRRCWWGYYY] is very important in determining the relative binding affinity of a given CS homology for wild-type or mutant p53. Our studies suggest that stereospecific alignment of the DNA-binding motifs within the CS may favor binding of wild-type p53 while misalignment may favor binding of mutant p53. Furthermore, we find that the maximum distances at which p53 DNA-binding CS homologies are functionally active vary for different sequences. Introduction of as few as 200 bp between one CS homology and the downstream TATA box can eliminate a 45-fold p53-mediated transactivation. We present evidence that the composition of the DNA which flanks a p53 DNA-binding consensus sequence may also modulate trans-activation.

Identification and antisense inhibition of a renin-angiotensin system in transgenic cardiomyocytes.

Cardiac myocytes (AT-1 cells) derived from heart tumors of mice transgenic for an atrial natriuretic factor promoter, SV40 large T-antigen DNA transgene, demonstrate properties consistent with normal cardiac myocytes but retain the capacity to proliferate in culture. We studied the renin-angiotensin system (RAS) and related growth regulation of these cells because AT-1 cells (or transgenically similar cells) may be useful to repair injured myocardium. This study reveals two separate and distinct findings: 1) AT-1 cells proliferate or hypertrophy in response to angiotensin II (ANG II), depending on their competence to proceed through the cell cycle; and 2) AT-1 cells possess components of a RAS, and angiotensinogen antisense experiments suggest that the RAS is functional in these cells. Specifically, AT-1 cells proliferate in response to ANG II in low-serum medium but hypertrophy in response to ANG II when first treated with mitomycin C (at a concentration that inhibits DNA replication but is not cytotoxic). The ANG II-mediated proliferative and hypertrophic responses are inhibited by DuP 753. In addition, there is a significant increase in the protein-to-DNA ratio of cells, which are proliferation-inhibited in the absence of ANG II treatment (20%, P < 0.05). DuP 753 also inhibits this hypertrophy, suggesting that these cells possess a functional RAS. AT-1 cells contain mRNAs for angiotensin-converting enzyme, renin, angiotensinogen, and the AT1 receptor as determined by sequence analysis of polymerase chain reaction amplification products. Antisense oligonucleotides complementary to the angiotensinogen mRNA specifically inhibit angiotensinogen mRNA accumulation and proliferation of AT-1 cells. In summary, these cells contain a growth-regulating RAS, suggesting that such a system may play a significant role in left ventricular hypertrophy.