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The NCI Pediatric Preclinical Testing Program (PPTP)
Cell Culture Laboratory

Director:    Min H. Kang, PharmD
Co-investigators:    C Patrick Reynolds, MD PhD

This laboratory is the cell culture (in vitro) portion of the NCI PPTP. The panel of cell lines includes neuroblastomas, rhabomyosarcomas, lymphomas, ALL, AML, ESFT, and brain tumor cell lines (see below for list of cell lines).

Methods employed:

The primary cytotoxicity assay for the lab is the DIMSCAN 96 well assay. DIMSCAN employs fluorescein diacetate (FDA) to identify viable cells, with quenching of fluorescence by eosin Y, and viable cells quantified using a DIMSCAN digital image microscopy system. (1,2). The DIMSCAN assay has a 4 log dynamic range when testing solid tumor (3, 4) and leukemia (5) cell lines Testing is carried out under standard culture conditions (20% O2), but drugs shown to be active in initial testing are also assessed in bone marrow-level oxygen tension (5 % O2) and in 2% O2, which approximates the oxygen levels found in many tumors (6). Assessing drug interactions (additivity, synergy, antagonism) is done employing fixed-ratios of drug concentrations and the combination index approach (4,7) All cell lines used are human, have been tested and shown to be free of mycoplasma, and have been demonstrated to be unique using short tandem repeats (8).

Laboratory Funding

NCI PPTP Grant

Laboratory Publications

  1. Whiteford CC, Bilke S, Greer BT, Braunschweig TA, Cenacchi N, Wei JS, Smith M, Houghton P, Reynolds CP, Lock R, Gorlick R, Khanna C, Thiele CJ, COG Phase 1 Consortium, Hewitt SM, Khan J: Credentialing preclinical pediatric xenograft models using gene expression and tissue microarray analysis. Cancer Research 67:32-40, 2007.
  2. Houghton PJ, Morton CL, Tucker C, Payne D, Favours E, Cole C, Gorlick R, Kolb EA, Zhang W, Lock R, Carol H, Tajbakhsh M, Reynolds CP, Maris JM, Courtright J, Keir ST, Friedman HS, Stopford C, Zeidner J, Wu J, Liu T, Billups CA, Khan J, Ansher S, Zhang J, Smith MA: The Pediatric Preclinical Testing Program: Description of models and early testing results. Pediatric Blood & Cancer 49:928-940, 2007.
  3. Maris JM, Courtright J, Houghton PJ, Morton CL, Gorlick R, Kolb EA, Lock R, Tajbakhsh M, Reynolds CP, Keir ST, Wu J, Smith MA: Initial testing (Stage 1) of the VEGFR inhibitor AZD2171 by the Pediatric Preclinical Testing Program. Pediatric Blood & Cancer 50:581-587, 2008.
  4. Houghton PJ, Morton CL, Tucker C, Gorlick R, Kolb EA, Zhang W, Lock R, Carol H, Reynolds CP, Keshelava N, Maris JM, Courtright J, Keir ST, Friedman HS, Stopford C, Wu J, Smith MA: Stage 1 testing of the proteasome inhibitor bortezomib by the Pediatric Preclinical Testing Program. Pediatric Blood & Cancer 50:37-45, 2008.
  5. Kolb EA, Gorlick R, Houghton PJ, Morton CL, Lock R, Tajbakhsh M, Reynolds CP, Maris JM, Keir ST, Billups CA, Smith MA: Initial testing of dasatinib by the Pediatric Preclinical Testing Program. Pediatric Blood & Cancer 50:1198-1206, 2008.
  6. Tajbakhsh M, Houghton PJ, Morton CL, Kolb EA, Maris JM, Keir ST, Wu J, Reynolds CP, Smith MA, Lock RB: Initial testing (Stage 1) of cisplatin by the Pediatric Preclinical Testing Program. Pediatric Blood & Cancer 50:992-1000, 2008.
  7. Houghton PJ, Morton CL, Kolb EA, Lock R, Reynolds CP, Maris JM, Keir ST, Billups CA, Tucker C, Smith MA: Initial testing (Stage 1) of the mTOR inhibitor rapamycin by the Pediatric Preclinical Testing Program. Pediatric Blood & Cancer 50:709-805, 2008.
  8. Lock R, Carol H, Houghton PJ, Morton CL, Kolb EA, Reynolds CP, Maris JM, Keir ST, Wu J, Smith MA: Initial testing (Stage 1) of the BH3 Mimetic ABT-263 by the Pediatric Preclinical Testing Program. Pediatric Blood & Cancer 50:1181-1189, 2008.
  9. Kolb EA, Gorlick R, Houghton PJ, Morton CL, Lock R, Carol H, Reynolds CP, Maris JM, Keir ST, Billups CA, Smith MA: Initial testing (stage 1) of a monoclonal antibody (SCH 717454) against the IGF-1 receptor by the pediatric preclinical testing program. Pediatr Blood & Cancer 50:1190-1197, 2008.
  10. Smith MA, Morton CL, Phelps DA, Kolb EA, Lock R, Carol H, Reynolds CP, Maris JM, Keir ST, Wu J, Houghton PJ. Stage 1 testing and pharmacodynamic evaluation of the HSP90 inhibitor alvespimycin (17-DMAG, KOS-1022) by the pediatric preclinical testing program. Pediatric Blood & Cancer 51(1):34-41, 2008.
  11. Maris JM, Courtright J, Houghton PJ, Morton CL, Kolb EA, Lock R, Tajbakhsh M, Reynolds CP, Keir ST, Wu J, Smith MA. Initial testing (stage 1) of sunitinib by the pediatric preclinical testing program. Pediatric Blood & Cancer 51:42-8, 2008.
  12. Neal G, Su X, Morton CL, Phelps D, Gorlick R, Lock RB, Reynolds CP, Maris JM, Friedman HS, Dome J, Khoury J, Triche TJ, Seeger RC, Gilbertson R, Khan J, Smith MA, Houghton PJ: Molecular characterization of the Pediatric Preclinical Testing panel. Clinical Cancer Research 14:4572-83, 2008.
  13. Morton C, Houghton PJ, Gorlick R, Kolb EA, Lock RB, Carol H, Keir ST, Reynolds CP, Kang MH, Maris JM, Billups C, Smith MA: Initial testing of aplidin by the pediatric preclinical testing program. Pediatr Blood & Cancer 53:509-12, 2009
  14. \Keshelava N, Houghton PJ, Morton CL, Lock RB, Keir ST, Maris JM, Reynolds CP, Gorlick R, Wu J, Smith MA: Initial testing (stage 1) of vorinostat (SAHA) by the Pediatric Preclinical Testing Program. Pediatr Blood & Cancer 53:505-8, 2009.
  15. Gorlick R, Kolb EA, Houghton PJ, Morton CL, Phelps D, Schaiquevich P, Stewart C, Keir ST, Lock R, Carol H, Reynolds CP, Maris JM, Wu J, Smith MA: Initial testing (stage 1) of lapatinib by the pediatric preclinical testing program. Pediatr Blood & Cancer 53:594-8, 2009
  16. Carol H, Lock R, Houghton PJ, Morton CL, Kolb EA, Gorlick R, Reynolds CP, Maris JM, Keir ST, Billups CA, Smith MA: Initial testing (stage 1) of the kinesin spindle protein inhibitor ispinesib by the pediatric preclinical testing program. Pediatr Blood & Cancer 24;53(7):1255-1263, 2009.
  17. Houghton PJ, Morton CL, Kang MH, Reynolds CP; Billups CA, Favours E, Payne-Turner D, Tucker C, Smith MA: Evaluation of cytarabine against Ewing sarcoma xenografts by the Pediatric Preclinical Testing Program. Pediatr Blood & Cancer (in press, 2009).
  18. Carol H, Houghton PJ, Morton CL, Kolb A, Gorlick R, Reynolds CP, Kang MH, Maris JM, Keir ST, Watkins A Smith MA, Lock RB: Initial testing of topotecan by the Pediatric Preclinical Testing Program. Pediatr Blood & Cancer 54(5):707-715, 2009.
  19. Smith MA, Morton CL,: Kolb, EA, Gorlick, R, Keir ST, Carol H, Lock RB, Kang MH, Reynolds CP, MD, Maris JM, Watkins AE, Houghton PJ: Initial testing (Stage 1) of mapatumumab (HGS-ETR1) by the Pediatric Preclinical Testing Program. Pediatr Blood & Cancer 54:307-10, 2010.
  20. Houghton PJ, Morton CL, Gorlick R, Lock RB, Carol H, Reynolds CP, Kang MH, Maris JM, Keir ST, Kolb EA, Wu J, Wozniak AW, Billups CA, Rubinstein L, Smith MA: Stage 2 combination testing of rapamycin with cytotoxic agents by the Pediatric Preclinical Testing Program. Mol Cancer Therapeutics 9:101-12, 2010.
  21. Maris JM, Morton CL, Gorlick R, Kolb EA, Lock R, Carol H, Keir ST, Reynolds CP, Kang MH, Wu J, Smith MA, Houghton PJ: Initial testing of the aurora kinase a inhibitor MLN8237 by the Pediatric Preclinical Testing Program (PPTP). Pediatr Blood & Cancer. 2010 Jan 27. [Epub ahead of print]
  22. Houghton PJ, Morton CL, Gorlick R, Kolb EA, Keir ST, Reynolds CP, Kang MH, Maris JM, Wu J, Smith MA: Initial testing of a monoclonal antibody (IMC-A12) against IGF-1R by the pediatric preclinical testing program. Pediatr Blood & Cancer. 2010 Feb 17. [Epub ahead of print].
  23. Kang MH, Smith MA, Morton CL, Nino Keshelava N, Houghton PJ, Reynolds CP: National Cancer Institute Pediatric Preclinical Testing Program: Model description for in vitro cytotoxicity testing. Pediatr Blood & Cancer (In Press, 2010).

Bibliography

  1. Proffitt RT, Tran JV, Reynolds CP: A fluorescence digital image microscopy system for quantitating relative cell numbers in tissue culture plates. Cytometry 24:204-213,1996.
  2. Keshelava N, Frgala T, .Krejsa J, Kalous O, Reynolds, CP: DIMSCAN: a microcomputer fluorescence-based cytotoxicity assay suitable for pre-clinical testing of combination chemotherapy. Methods in Molecular Medicine Chemosensitivity Vol 1 ed. Blumenthal RD, Totowa: Humana Press pp 139-154, 2005.
  3. Keshelava N, Seeger RC, Groshen S, Reynolds CP: Drug resistance patterns of human neuroblastoma cell lines derived from patients at different phases of therapy. Cancer Research 58:5396-5405, 1998.
  4. Maurer BJ, Cabot MC, Reynolds CP: Syngergism of N-(4-hydroxypheynl)retinamide cytotoxcity by modulators of ceramide metabolism in solid tumor cell lines. J Natl Cancer Inst 92:1897-1908, 2000.
  5. O'Donnell PH, Guo WX, Reynolds CP, Maurer BJ: N-(4-hydroxyphenyl)retinamide increases ceramide and Is cytotoxic to acute lymphoblastic leukemia cell lines, but not to non-malignant lymphocytes. Leukemia 16:902-910, 2002.
  6. Grigoryan R, Keshelava N, Anderson C, Reynolds, CP: In vitro testing of chemosensitivity in physiological hypoxia. Methods in Molecular Medicine Chemosensitivity Vol 1 ed. Blumenthal RD, Totowa: Humana Press pp 87-100, 2005.
  7. Reynolds, CP, Maurer BJ: Assessing response to anti-neoplastic drug combinations in tissue culture models. Methods in Molecular Medicine Chemosensitivity Vol 1 ed. Blumenthal RD, Totowa: Humana Press pp 173-184, 2005.
  8. Frgala T, Kalous O, Proffitt RT, Reynolds CP: A novel cytotoxicity assay with a 4 log dynamic range that identifies synergistic drug combinations. Molecular Cancer Therapeutics 6:886-89, 2007.

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