Abstract
Specific recurring chromosomal translocations and deletions are found in a variety of cancers. In hematopoietic malignancies, many of these chromosomal aberrations result from mistakes involving V(D)J recombination. V(D)J recombination is required for the formation of functional T-cell receptor genes in T-cells and antibody genes in B-cells. This is an inherently dangerous process, however, because double-strand breaks are introduced into the chromosomes. Molecular evidence indicates that failure of the fidelity of this process results in the activation of proto-oncogenes or the inactivation of tumor suppressor genes. Here we describe sensitive, quantitative PCR assays for the measurement of such events in human lymphocytes. One assay measures the frequency of t(14;18) translocations that result in the dysfunctional regulation of the anti-apoptotic gene BCL-2. The other assay measures the frequency of a deletion caused by illegitimate V(D)J recombination in the X-linked HPRT gene.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lipford E, Wright JJ, Urba W et al (1987) Refinement of lymphoma cytogenetics by the chromosome 18q21 major breakpoint region. Blood 70:1816–1823
Yunis JJ, Oken MM, Kaplan ME, Ensrud KE, Howe RR, Theologides A (1982) Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin’s lymphomas. N Engl J Med 307:1231–1236
Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348:334–336
Crescenzi M, Seto M, Herzig GP, Weiss PD, Griffith RC, Korsmeyer SJ (1988) Thermostable DNA polymerase chain reaction amplification of t(14;18) chromosome breakpoints and detection of minimal residual disease. Proc Natl Acad Sci U S A 85:4869–4873
Ngan BY, Nourse J, Cleary ML (1989) Detection of chromosomal translocation t(14;18) within the minor cluster region of bcl-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas. Blood 73:1759–1762
Cotter F, Price C, Zucca E, Young BD (1990) Direct sequence analysis of the 14q+ and 18q- chromosome junctions in follicular lymphoma. Blood 76:131–135
Eick S, Krieger G, Bolz I, Kneba M (1990) Sequence analysis of amplified t(14;18) chromosomal breakpoints in B-cell lymphomas. J Pathol 162:127–133
Liu Y, Hernandez AM, Shibata D, Cortopassi GA (1994) BCL2 translocation frequency rises with age in humans. Proc Natl Acad Sci U S A 91:8910–8914
Zhang XY, Ehrlich M (1994) Detection and quantitation of low numbers of chromosomes containing bcl-2 oncogene translocations using semi-nested PCR. BioTechniques 16:502–507
Fuscoe JC, Setzer RW, Collard DD, Moore MM (1996) Quantification of t(14;18) in the lymphocytes of healthy adult humans as a possible biomarker for environmental exposures to carcinogens. Carcinogenesis 17:1013–1020
Fuscoe JC, Zimmerman LJ, Lippert MJ, Nicklas JA, O’Neill JP, Albertini RJ (1991) V(D)J recombinase-like activity mediates hprt gene deletion in human fetal T-lymphocytes. Cancer Res 51:6001–6005
Fuscoe JC, Zimmerman LJ, Harrington-Brock K et al (1992) V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans. Mutat Res 283:13–20
Fuscoe JC, Vira LK, Collard DD, Moore MM (1997) Quantification of hprt gene deletions mediated by illegitimate V(D)J recombination in peripheral blood cells of humans. Environ Mol Mutagen 29:28–35
Chen CL, Fuscoe JC, Liu Q, Relling MV (1996) Etoposide causes illegitimate V(D)J recombination in human lymphoid leukemic cells. Blood 88:2210–2218
Chen CL, Fuscoe JC, Liu Q, Pui CH, Mahmoud HH, Relling MV (1996) Relationship between cytotoxicity and site-specific DNA recombination after in vitro exposure of leukemia cells to etoposide. J Natl Cancer Inst 88:1840–1847
Chen CL, Woo MH, Neale GA et al (1998) A human lymphoid leukemia cell line with a V(D)J recombinase-mediated deletion of hprt. Mutat Res 403:113–125
Fuscoe JC, Knapp GW, Hanley NM et al (1998) The frequency of illegitimate V(D)J recombinase-mediated mutations in children treated with etoposide-containing antileukemic therapy. Mutat Res 419:107–121
Scheerer JB, Xi L, Knapp GW, Setzer RW, Bigbee WL, Fuscoe JC (1999) Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults. Mutat Res 431:291–303
Edwards A, Voss H, Rice P et al (1990) Automated DNA sequencing of the human HPRT locus. Genomics 6:593–608
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Fuscoe, J.C. (2020). Simultaneous Quantification of t(14;18) and HPRT Exon 2/3 Deletions in Human Lymphocytes. In: Keohavong, P., Singh, K., Gao, W. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 2102. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0223-2_21
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0223-2_21
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0222-5
Online ISBN: 978-1-0716-0223-2
eBook Packages: Springer Protocols