L. Michael Carastro

Role of p73 Dinucleotide Polymorphism in Prostate Cancer and p73 Protein Isoform Balance.

Background. Molecular markers for prostate cancer (PCa) risks are currently lacking. Here we address the potential association of a dinucleotide polymorphism (DNP) in exon 2 of the p73 gene with PCa risk/progression and discern any disruption of p73 protein isoforms levels in cells harboring a p73 DNP allele. Methods. We investigated the association between p73 DNP genotype and PCa risk/aggressiveness and survival by fitting logistic regression models in 1,292 incident cases and 682 controls. Results. Although we detected no association between p73 DNP and PCa risk, a significant inverse relationship between p73 DNP and PCa aggressiveness (AT/AT + GC/AT versus GC/GC, OR = 0.55, 95%Cl = 0.31–0.99) was detected. Also, p73 DNP is marginally associated with overall death (dominant model, HR = 0.76, 95%Cl = 0.57–1.00, P = 0.053) as well as PCa specific death (HR = 0.69, 95%Cl = 0.45–1.06, P = 0.09). Western blot analyses for p73 protein isoforms indicate that cells heterozygous for the p73 DNP have lower levels of ∆Np73 relative to TAp73 (P < 0.001). Conclusions. Our findings are consistent with an association between p73 DNP and low risk for PCa aggressiveness by increasing the expressed TAp73/∆Np73 protein isoform ratio.

Corrigendum to “A novel SNP in a vitamin D response element of the CYP24A1 promoter reduces protein binding, transactivation, and gene expression” [J. Steroid Biochem. Mol. Biol. 112 (2008) 47–54]

The authors realized after the publication of the above article that the numbering used to indicate the positions of the variants and response elements relative to the transcription start site were not accurate. The correct positions for the eight polymorphisms described in Table 1 are one nucleotide further from the start site, i.e. −104, −128, −227, etc. The novel variants have since been submitted to NCBI with the correct annotation. In addition, the positions of the promoter elements shown in Figure 1 should be −290 to −276 (VDRE2) and −170 to−147 (VDRE1). We apologize for any inconvenience or confusion this may have caused the readers. Dr. Wilson regrets that during publication, Dr. Carastro was not included as an author. The corrected author list is published above.

Assessing stakeholder perceptions of the american society for biochemistry and molecular biology accreditation program for baccalaureate degrees: Assessing Stakeholder Perceptions

The American Society for Biochemistry and Molecular Biology (ASBMB) began an accreditation program in 2013. The criteria for accreditation of undergraduate programs include sufficient infrastructure ‐ number and expertise of faculty, physical space and equipment, support for faculty and students ‐ and incorporation of core concepts in the curriculum ‐ structure and function of biomolecules; information storage; energy transfer; and quantitative skills. Students in accredited programs are able to have their degrees ASBMB certified by taking an exam focused on knowledge or skills across the four core concept areas. Members of the accreditation committees administered a survey to key stakeholders in the BMB community: undergraduate programs, both those that have applied for accreditation and those that have not; alumni/ae of accredited programs; graduate and professional programs; and employers. The goals of the study were to gauge the success of the program and determine necessary areas of improvement. The results indicate that the major benefits of applying for accreditation are the impetus to gather data and analysis not generally collected, and access to assessment data via the exam. However, stakeholders outside of the undergraduate community showed little awareness of the accreditation program. Additionally, the application process itself was seen to be very time consuming. This feedback will be used to improve the process and engage in further outreach.

Abstract 3003: p73 DNP genotype influences effects of etoposide and 5-aza-2’-deoxycytidine on p73 mRNA isoforms ratios in human cancer cell lines

Background: The p73 gene is a member of the p53 tumor suppressor family. A dinucleotide polymorphism (DNP) in p73 is a G4C14-to-A4T14 linked pair of transitions located in exon 2, between the P1 and P2 promoters, from which TAp73 and DeltaNp73 (DNp73) mRNA isoforms are transcribed, respectively. A p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14-to-A4T14 linked pair of transitions located in exon 2 and lies between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and deltaNp73 (DNp73) isoforms, respectively. These p73 mRNA isoforms, TAp73 and αNp73, encode protein isoforms which differ in their N-termini. TAp73 isoforms include the full-length N-terminal sequence and are transcriptionally active. αNp73 isoforms lack the N-terminal trans-activation domain and are dominate negative. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI = 0.31-0.99] for aggressive prostate cancer and (2) increased TAp73/DNp73 protein isoform ratios in ten human cancer cell lines. Etoposide is known to induce p73 gene expression. Others recently reported treatment of T-47D cells with 5-aza-2’-deoxycytidine (5AZA) resulted in increased TAp73/DNp73 mRNA ratios. Hypothesis: We hypothesized that treatment with 5AZA increases the TAp73/DNp73 mRNA ratios in human cell lines and that the presence of the p73 DNP allele potentiates 5AZA-induced increases TAp73/DNp73 cellular ratios. Methods. We treated human cancer cell lines with 50 microM etoposide for 24 hrs and/or 20 microM 5AZA for 48 hrs. RNA was isolated, converted to cDNA and used in TaqMan RT-PCR assays to detect total p73, TAp73 or αNp73 mRNAs. Results: Cell lines wild-type for p73 DNP (DU145, T47-D, MDA-MB-468) had TAp73/DNp73 mRNA ratios that were lower than those heterozygous for p73 DNP (PC-3 and MDA-MB-231) following co-treatment with 5AZA and etoposide or with etoposide alone (p Conclusion: Of the five cell lines used in this study, cells that harbor a p73 DNP allele had the highest TAp73/DNp73 mRNA ratios following co-treatment with 5AZA and etoposide or with etoposide alone as compared to wild-type cell p73 DNP, which is consistent with our previously reported data. Lastly, our data in this work assessing the effect of 5AZA treatment of T47-D cells on TAp73/DNp73 mRNA ratios is not consistent with a previously reported study. Citation Format: Zachary Connelly, L. Michael Carastro. p73 DNP genotype influences effects of etoposide and 5-aza-2’-deoxycytidine on p73 mRNA isoforms ratios in human cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3003.

Abstract 5569: p73 dinucleotide polymorphism genotyping of human cancer cell lines

The p73 gene is a member of the p53 tumor suppressor family. The p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14-to-A4T14 linked pair of transitions located in exon 2 between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and ΔNp73 isoforms, respectively. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI = 0.31-0.99] for aggressive prostate cancer (PCa) and (2) increased TAp73/ΔNp73 protein isoform ratios in ten human cancer cell lines. We hypothesize the presence of the p73 DNP allele causes altered p73 promoter usage resulting in increased TAp73/ΔNp73 protein isoform ratios, which could explain the observed decreased risk for PCa aggressiveness. Our ultimate goal is to assess the potential of p73 DNP as a biomarker for lower aggressive PCa risk. Therefore, our initial aim in this study was to determine the p73 DNP genotype in ten additional human cancer cell lines (DU 145, JEG-3, K-562, LNCaP, MDA-MB-231, MDA-MB-468, MRC-5, PC-3, SW48 and U-2OS) not yet characterized for p73 DNP status. We previously reported the p73 DNP genotype of three human cancer cell lines that we used as genotyping controls: Caco-2 (homozygous polymorphic), NCI-H1299 (homozygous wild type), and HepG2 (heterozygous). Cell lines were cultured and genomic DNA was isolated and quantitated. Cell line genomic DNA samples were used in TaqMan Real Time-PCR allelic discrimination assays to determine the p73 DNP genotypes. Our data conclusively determined that DU 145, JEG-3, K-562, LNCaP, MDA-MB-468, MRC-5, SW48 and U-2OS were p73 DNP homozygous wild type, while PC-3 and MDA-MB-231 werw p73 DNP heterozygous. Citation Format: L. Michael Carastro, Kaia K. Hampton, Ricardo A. Cordova Estupinan, Hyun Y. Park, Dongwha Kim, Jong Y. Park. p73 dinucleotide polymorphism genotyping of human cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5569. doi:10.1158/1538-7445.AM2015-5569

Abstract 5070: Role of p73 di-nucleotide polymorphism in prostate cancer and p73 protein isoform balance

The p73 gene is a member of the p53 tumor suppressor family. Compared to p53, the molecular consequences of and risk factors associated with variants of the p73 gene are not well understood. Prostate cancer (PCa) is the most frequently occurring cancer in men, and the second leading cause of cancer mortality among US men. Available molecular markers for PCa risk of incidence or aggressiveness are currently lacking. Though the mechanism of PCa progression is not well understood, several lines of evidence support a role for a di-nucleotide polymorphism (DNP) in the p73 gene as a risk factor for several cancer types. However, results are inconsistent. We investigated a DNP linked pair of transition changes, G4C14-to-A4T14 (rs1801173), located in the 5′-UTR portion of exon 2 of the p73 gene. The goals of this study were to address the potential associations between p73 DNP (rs1801173) and PCa risk and/or progression, and to discern any detectable disruption of p73 protein isoforms in cells harboring the p73 DNP allele. We have analyzed 1,292 PCa patients and 682 healthy control men. Although we detected no association between p73 DNP and PCa risk (OR =1.02, 95%CI=0.86-1.21), a significant inverse relationship between p73 DNP and PCa aggressiveness (OR = 0.55, 95%CI=0.31-0.99) was detected. Several p73 protein isoforms have been identified and can be grouped into two major categories, TAp73 and ΔNp73, which differ in their N-termini and are transcribed from different promoters. The transcriptionally active TAp73 isoforms are transcribed from the P1 promoter and include the full-length N-terminal sequence encompassing exons 1 through 3. However, the ΔNp73 isoforms are transcribed from promoter P2 and do not contain the N-terminal trans-activation (TA) domain. Therefore, the ΔNp73 protein isoform is dominate negative toward TAp73 because ΔNp73 isoforms are able to form tetramers with TAp73, as well as p53, but do not activate transcription of p73- or p53-target genes. The balance between TAp73 and ΔNp73 is disrupted in many cancer types. In this study, Western Blot analyses of eleven cancer cell lines for p73 protein isoforms indicate that the two cell lines with heterozygous genotype for p73 DNP have higher TAp73/ΔNp73 ratios (TAp73/ΔNp73 = 1.39 and 1.4) than the other wild-type cell lines not harboring the p73 DNP allele (TAp73/ΔNp73 = 0.68 to 0.97). Our combined findings are consistent with an association between the p73 DNP allele and lower risk for PCa aggressiveness by increasing the expressed TAp73/ΔNp73 protein isoform ratio. Citation Format: L. Michael Carastro, Hui-Yi Lin, Hyun Y. Park, Donghwa Kim, Selina Radlein, Kaia K. Hampton, Ardeshir Hakam, Babu Zachariah, Julio M. Pow-Sang, Jong Y. Park. Role of p73 di-nucleotide polymorphism in prostate cancer and p73 protein isoform balance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5070. doi:10.1158/1538-7445.AM2014-5070