Single-cell analysis of p16(INK4a) and p21(WAF1) expression suggests distinct mechanisms of senescence in normal human and Li-Fraumeni Syndrome fibroblasts.

Herein we used single-cell observation methods to gain insight into the roles of p16(INK4A) and p21(WAF1) (hereafter p16 and p21) in replicative senescence and ionizing radiation-induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li-Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence-associated beta-galactosidase. In addition, proliferating early-passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53-proficient (normal and AT) fibroblasts. In p53-deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation-triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild-type p53 activity. The possibility that one of the tumor-suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results.

Effects of mutations involving cysteine residues distal to the S281HCC motif at the C-terminus on the functional characteristics of a truncated ectodomain-only thyrotropin receptor anchored on glycosylphosphatidyl-inositol.

BACKGROUND: Cysteine (Cys) residues pair to form disulfide bonds that are important in maintaining structure and function of the thyrotropin receptor (TSHR). There are 11 Cys residues in the ectodomain (ECD). Cys 41 at the N-terminus and Cys 283 at the SHCC motif have been identified as important for ligand binding. The present study evaluated the effects of mutating Cys distal to the S281HCC motif at the C-terminus of the ECD on the functional characteristics of TSHR. METHODS: We introduced (i) individual Cys and (ii) consecutive cumulative Cys mutations into the starting template SHCS-TSHR, a truncated TSHR-ECD moiety previously shown to behave like the wild-type TSHR. Each mutant receptor was evaluated for relative specific binding (RSB), calculated as a measure of TSH-binding ability after normalization with receptor surface expression. RESULTS: In the first approach, RSB was severely affected when Cys 390 and Cys 398 were individually switched to serine. Failed receptor trafficking occurred with Cys 408 mutation. These findings were likely results of altered receptor conformation due to illegitimate disulfide bridge formation. Only SHCS-301 TSHR bound TSH in a specific manner, and it formed the base for sequential Cys mutations. Through this second approach, both Cys 301 and 390 could be removed simultaneously without hindering TSH binding significantly. Cys 398, however, was shown to be critical. Its absence resulted in huge loss of TSH binding. Leaving Cys 283 and 398 as the only Cys pair in the C-terminus alone could support 40% of the total ligand-binding capacity. CONCLUSIONS: From these data, we proposed Cys 398 as a stable disulfide bond partner of Cys 283, corroborating with a model based on evolutionary history of TSHR across species. This pairing of Cys 283 and Cys 398 also provides an objective alternative to conventional hypotheses on Cys coupling based on other predictive models.

Increased but error-prone nonhomologous end joining in immortalized lymphoblastoid cell extracts from adult cancer patients with late radionecrosis.

PURPOSE: To study nonhomologous end joining in extracts of two lymphoblastoid cell lines derived from patients with late radionecrosis after radiotherapy. Both cell lines were previously shown to exhibit impaired rejoining of DNA double-strand breaks in a pulse-field gel electrophoresis assay. METHODS AND MATERIALS: We used a cell-free system and quantitative real-time polymerase chain reaction, as well as sequencing analysis of end joining products. RESULTS: Paradoxically, extracts of the two cell lines display increased rates of in vitro end joining of noncohesive termini compared with normal cell extracts. This increase was seen in the absence of added deoxyribonucleoside triphosphates and was sensitive to inhibition by wortmannin. Sequencing of the joined products revealed that, despite increased rates of end joining, the process was error prone with a greater frequency of deletions compared with that observed in normal controls. CONCLUSION: These findings are consistent with the suggestion that a promiscuous, deletion-prone abnormality of nonhomologous end joining might underpin the predisposition of certain radiotherapy patients to late radionecrosis. We hypothesize that some individuals might harbor subclinical defects in nonhomologous end joining that clinically manifest on challenge with high-dose radiation. Because both quantitative and qualitative aspects of end joining have demonstrably been influenced, we recommend that the study of patient samples should involve a combination of quantitative methods (e.g., quantitative real-time polymerase chain reaction), sequencing analysis, and a comparison of multiple join types.

Aberrant sensing of extracellular Ca2+ by cultured ataxia telangiectasia fibroblasts.

Ataxia telangiectasia (AT) is a human hereditary syndrome whose underlying gene product, ataxia telangiectasia mutated (ATM) protein kinase, is involved in multiple intracellular signaling pathways. We demonstrated previously that AT fibroblasts are defective in intracellular Ca(2+) mobilization in response to both stress-inducing and mitogenic stimuli. To extend these findings, normal and AT cells were exposed to serum in the presence of different concentrations of extracellular Ca(2+) ([Ca(2+)](o)), and release of intracellular Ca(2+), activation of calmodulin-dependent protein kinase II and phosphorylation of kinases ERK1 and 2 were monitored. When maintained in high [Ca(2+)](o) (0.42 mM), normal fibroblasts responded to serum introduction more rapidly and efficiently than did AT cells. Unexpectedly, decreasing the [Ca(2+)](o) in the medium had a diametrically opposite effect. Under low [Ca(2+)](o) (0.0022 mM) conditions, normal cells were slow and inefficient in their responses, whereas AT cells showed a substantial improvement in all three end points. These findings demonstrate that loss of ATM kinase function deregulates the extracellular calcium-sensing receptor (CaR). This malfunction presumably arises from a post-transcriptional event, since CaR mRNA proved to be normal in AT cells. Together, our data suggest that ATM may mediate cell response to mitogenic factors by tightly regulating the set point of the CaR and thereby modulating the crosstalk between this metabotropic receptor and growth factor receptors. Alternatively, the faulty sensing of extracellular calcium in AT cells may be secondary to a state of chronic oxidative stress attributable to ATM deficiency.

Substrate specificity and sequence preference of G:T mismatch repair: incision at G:T, O6-methylguanine:T, and G:U mispairs in DNA by human cell extracts.

Extracts of two human glioma cell lines (lacking O6-methylguanine DNA-methyltransferase) (i.e., A1235 and its alkylation-resistant derivative A1235-MR4) were examined for their ability to execute strand incision at different base mismatches in model (45-bp) DNA. These heteroduplex substrates were of the same sequence except for the presence, at the same site, of one of three mispairs: G:T, O6-methylguanine:T (m6G:T), and G:U. The parental (A1235) extract, when supplemented with ATP and human thymine DNA glycosylase (TDG), acted proficiently on all three substrates, incising immediately 5' to the mismatched thymine or uracil residue. In contrast, the derivative extract, under the same conditions, recognized only the G:U substrate. The activity of the A1235 extract toward the G:T (or m6G:T) substrate was markedly reduced in the absence of ATP, whereas the G:U substrate was incised rapidly by both extracts irrespective of the addition of ATP. These combined data confirm and extend our earlier findings demonstrating that human cells possess two G:T incision activities, one efficient and ATP-dependent and the other inefficient and ATP-independent. The derivative extract lacks the former activity but retains the latter activity. In substrate competition assays, the G:U substrate inhibited the ATP-dependent G:T incision activity to a greater extent than did the G:T substrate itself. Given the well-known substrate preference of TDG for G:U as compared to G:T, this unexpected result implies that TDG may be an integral component of the ATP-dependent G:T incision machinery in human cells. Finally, the base 5' to the mismatched G in the G:T mispair conferred sequence preference on the A1235 extract in the presence of ATP and TDG, with a pyrimidine (especially cytosine) being much favored over a purine. This latter observation suggests that the ATP-dependent G:T incision activity is designed to repair deaminated 5-methycytosine lesions in CpG islands, the methylation of which is linked to control of gene expression.

Gene therapy of melanoma pulmonary metastasis by intramuscular injection of plasmid DNA encoding tissue inhibitor of metalloproteinases-1.

Tumor cell invasion and metastasis are a complex multistep process that involves the degradation of extracellular matrix proteins by matrix metalloproteinases. Tissue inhibitor of metalloproteinase-1 (TIMP-1) acts as a negative regulator of matrix metalloproteinases and thus prevents tumor cell invasion and metastasis by preserving extracellular matrix integrity. In the present study, we investigated whether increasing serum TIMP-1 levels by gene transfer would decrease experimental pulmonary metastasis of melanoma in C57BL/6 mice. Female animals bearing B16F10 melanoma pulmonary metastasis were injected intramuscularly twice per week with 100 microg of plasmid DNA encoding the human TIMP-1 cDNA (TIMP-1pDNA). Substantive levels of serum human TIMP-1 were observed 3 days after single injection and were found for 6 days thereafter. Pulmonary metastasis was significantly reduced in the mice following 4 weeks of TIMP-1 treatment as compared to the controls that were treated with the plasmid DNA vector alone. Further reduction of pulmonary metastasis and increase in survival were realized by intraperitoneal injection of 1000 U of IL-2 twice per week in combination with TIMP-1 treatment. In a parallel in vitro study, a 3-fold increase in TIMP-1 expression was observed in NIH3T3 cells after IL-2 treatment. Therefore, up-regulation of TIMP-1 expression by IL-2 likely contributed to the additive effect of IL-2 and TIMP-1 in reducing metastatic disease in the animal model. In conclusion, our findings support the potential of TIMP-1 gene therapy for the prevention of metastatic melanoma.