Cyclosporin a inhibits calcineurin/nuclear factor of activated T-cells signaling and induces apoptosis in retinoblastoma cells.

PURPOSE: Although the clinical efficacy of cyclosporin A (CSA) in retinoblastoma (RB) has been attributed to multidrug resistance reversal activity, the authors hypothesized that CSA is also directly toxic to RB cells through inhibition of calcineurin (CN)/nuclear factor of activated T-cells (NFAT) signaling. METHODS: Antiproliferative effects of CSA, PSC-833 (a CSA analogue that does not inhibit CN), and FK506 (a CN inhibitor structurally unrelated to CSA) were evaluated in Y79 and Weri-RB1 cells by WST-1 assay. Apoptosis induction by CSA and PSC-833 was measured by detection of caspase 3/7 activity and by flow cytometry, using annexin-V and 7-AAD stains. Expression of CN was assayed in RB cells by immunocytochemistry. Expression of NFAT, a CN-dependent transcription factor family, and FK506 binding protein 12/12.6 (FKBP12/12.6), effectors of CN inhibition by FK506, was assayed in RB cells by Western blot analysis. NFAT activity was assayed in CSA-treated and -untreated Y79 cells transfected with an NFAT-sensitive reporter gene. RESULTS: CSA induced dose-dependent antiproliferative and proapoptotic effects at clinically achievable levels in Y79 and Weri-RB1 cells. PSC-833 induced antiproliferative effects only at nonphysiologic concentrations with minimal associated apoptosis. FK506 induced minimal antiproliferative effects in RB cell lines, probably due to trace or absent FKBP12/12.6 expression. RB cell lines expressed CN-alpha, CN-beta, NFATc1, and NFATc3. CSA treatment also potently inhibited NFAT-mediated reporter gene transcription. CONCLUSIONS: These results demonstrate functional integrity of the CN/NFAT signaling cascade in RB cells and suggest that CSA is cytotoxic to RB cells through inhibition of this pathway and consequent apoptosis induction.

Rapid identification of germline mutations in retinoblastoma by protein truncation testing.

OBJECTIVE: To demonstrate the utility of protein truncation testing (PTT) for rapid detection and sequencing of germline mutations in the retinoblastoma tumor suppressor gene (RB1). METHODS: We performed PTT, a technique based on the in vitro synthesis of protein from amplified RNA, on 27 probands from 27 kindreds with hereditary retinoblastoma. In 4 kindreds, PTT was also performed on 1 additional affected relative. Ten unrelated patients without retinoblastoma were included as negative control subjects. All PTT-detected mutations were further analyzed by focused sequencing of genomic DNA. When no mutation was detected by PTT, we performed exon-by-exon sequencing, as well as cytogenetic analysis by Giemsa-trypsin-Giemsa banding and by fluorescent in situ hybridization for RB1. The results of proband testing were used for direct genetic testing by polymerase chain reaction and sequencing in 11 relatives from 7 of the 27 kindreds. RESULTS: Of the probands tested, 19 (70%) of 27 tested positive for germline mutations by PTT. In 1 kindred, the proband had negative PTT results but an additional affected relative had positive PTT results. Focused DNA sequencing of 1 patient with positive PTT results from each of the 20 kindreds with positive PTT results revealed truncating mutations in 19 kindreds. Four demonstrated frameshift deletions, 6 had splice site mutations, and 9 showed nonsense mutations. Further analysis by genomic exon-by-exon sequencing and karyotype analysis of the 8 probands who tested negative for germline mutations by PTT revealed 1 splice site mutation, 2 missense mutations, and 1 chromosomal deletion. Focused sequencing based on positive PTT results was successfully used to confirm shared truncating mutations in additional affected family members in 2 kindreds. Using a multitiered approach to genetic testing, 23 (85%) of 27 kindreds had mutations identified and those detected by PTT received a positive result in as few as 7 days. In control subjects, PTT produced no false-positive results. CONCLUSIONS: Protein truncation testing is an effective, rapid single-modality screen for germline mutations in patients with retinoblastoma. When used as an initial screen, PTT can increase the yield of additional testing modalities, such as sequencing and chromosomal analysis, providing a timely and cost-effective approach for the diagnosis of heritable germline mutations in patients with retinoblastoma.Clinical Relevance The clinical application of PTT in retinoblastoma will improve detection of germline retinoblastoma mutations, which will supply critical information for prognosis, treatment planning, follow-up care, and genetic counseling.