BTK as a mediator of radiation-induced apoptosis in DT-40 lymphoma B cells

Science

Volumn 273, Issue 5278, 1996, Pages 1096-1100

  Uckun, Fatih M ^a   Waddick, Kevin G ^a   Mahajan, Sandeep ^a   Jun, Xiao ^a   Takata, Minoru ^b   Bolen, Joseph ^c   Kurosaki, Tomohiro ^b  

^a UNIVERSITY OF MINNESOTA   (United States)

^b YALE UNIVERSITY SCHOOL OF MEDICINE   (United States)

^c MERCK RESEARCH LABORATORIES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN TYROSINE KINASE;

ANIMAL CELL; APOPTOSIS; ARTICLE; CELL DEATH; CHICKEN; CONTROLLED STUDY; DNA DAMAGE; ENZYME ACTIVITY; ENZYME DEFICIENCY; ETIOLOGY; FLOW CYTOMETRY; GENETIC RECOMBINATION; LYMPHOMA CELL; LYMPHOMA CELL LINE; NONHUMAN; PRIORITY JOURNAL; RADIATION INJURY; RADIOSENSITIVITY;

ANIMALIA; GALLUS GALLUS;

EID: 0029838226     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5278.1096     Document Type: Article

References (84)

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31. The predominant PTKs that are abundantly expressed in B lineage lymphoid cells include the TEC PTK family member BTK (3, 4, 15, 16), the cytoplasmic PTK SYK (3, 4, 6, 7), and the SRC PTK family member LYN (3-5). DT-40 cells express high levels of BTK, SYK, and LYN kinases, whereas SRC, LCK, FYN, BLK, YES, and HCK kinases are not expressed at detectable levels. Stimulation of wild-type as well as BTK-deficienl DT-40 cells with M4 anti-IgM (1 to 4 μ9/ml) induces tyrosine phosphorylation of multiple substrates (M. Takata and T. Kurosaki, unpublished observations). In wild-type DT-40 cells, anti-IgM stimulation activates LYN, SYK, and BTK kinases (9, 10) [M. Takata et al., EMBO J. 13, 1341 (1994); K Nagai, M. Takata, H, Yamamura, T. Kurosaki, J. Biol. Chem. 270, 6824 (1995); D. J. Rawlings et al., Science 271, 822 (1996)].
32. The predominant PTKs that are abundantly expressed in B lineage lymphoid cells include the TEC PTK family member BTK (3, 4, 15, 16), the cytoplasmic PTK SYK (3, 4, 6, 7), and the SRC PTK family member LYN (3-5). DT-40 cells express high levels of BTK, SYK, and LYN kinases, whereas SRC, LCK, FYN, BLK, YES, and HCK kinases are not expressed at detectable levels. Stimulation of wild-type as well as BTK-deficienl DT-40 cells with M4 anti-IgM (1 to 4 μ9/ml) induces tyrosine phosphorylation of multiple substrates (M. Takata and T. Kurosaki, unpublished observations). In wild-type DT-40 cells, anti-IgM stimulation activates LYN, SYK, and BTK kinases (9, 10) [M. Takata et al., EMBO J. 13, 1341 (1994); K Nagai, M. Takata, H, Yamamura, T. Kurosaki, J. Biol. Chem. 270, 6824 (1995); D. J. Rawlings et al., Science 271, 822 (1996)].
33. The predominant PTKs that are abundantly expressed in B lineage lymphoid cells include the TEC PTK family member BTK (3, 4, 15, 16), the cytoplasmic PTK SYK (3, 4, 6, 7), and the SRC PTK family member LYN (3-5). DT-40 cells express high levels of BTK, SYK, and LYN kinases, whereas SRC, LCK, FYN, BLK, YES, and HCK kinases are not expressed at detectable levels. Stimulation of wild-type as well as BTK-deficienl DT-40 cells with M4 anti-IgM (1 to 4 μ9/ml) induces tyrosine phosphorylation of multiple substrates (M. Takata and T. Kurosaki, unpublished observations). In wild-type DT-40 cells, anti-IgM stimulation activates LYN, SYK, and BTK kinases (9, 10) [M. Takata et al., EMBO J. 13, 1341 (1994); K Nagai, M. Takata, H, Yamamura, T. Kurosaki, J. Biol. Chem. 270, 6824 (1995); D. J. Rawlings et al., Science 271, 822 (1996)].
34. The predominant PTKs that are abundantly expressed in B lineage lymphoid cells include the TEC PTK family member BTK (3, 4, 15, 16), the cytoplasmic PTK SYK (3, 4, 6, 7), and the SRC PTK family member LYN (3-5). DT-40 cells express high levels of BTK, SYK, and LYN kinases, whereas SRC, LCK, FYN, BLK, YES, and HCK kinases are not expressed at detectable levels. Stimulation of wild-type as well as BTK-deficienl DT-40 cells with M4 anti-IgM (1 to 4 μ9/ml) induces tyrosine phosphorylation of multiple substrates (M. Takata and T. Kurosaki, unpublished observations). In wild-type DT-40 cells, anti-IgM stimulation activates LYN, SYK, and BTK kinases (9, 10) [M. Takata et al., EMBO J. 13, 1341 (1994); K Nagai, M. Takata, H, Yamamura, T. Kurosaki, J. Biol. Chem. 270, 6824 (1995); D. J. Rawlings et al., Science 271, 822 (1996)].
35. 137Cs irradiator; J. L. Shephard, Glendale, CA, Model Mark I) with 4 or 8 Gy γ-irradiation at a dose rate of 1 Gy/min under aerobic conditions as described [F. M. Uckun et al., Cancer Res. 53, 1431 (1993); F. M. Uckun et al., J Clin. Invest. 91, 1044 (1993); F. M. Uckun et al., J. Biol. Chem. 271, 6389 (1996)], Before irradiation, cells were sometimes incubated with the tyrosine kinase inhibitor genistein (30 μg/ml for 1 hour) at 37°C to examine the role of PTK in radiation-induced apoptosis as described (2) [F. M. Uckun et al., ibid. 90, 252 (1993)].
36. 137Cs irradiator; J. L. Shephard, Glendale, CA, Model Mark I) with 4 or 8 Gy γ-irradiation at a dose rate of 1 Gy/min under aerobic conditions as described [F. M. Uckun et al., Cancer Res. 53, 1431 (1993); F. M. Uckun et al., J Clin. Invest. 91, 1044 (1993); F. M. Uckun et al., J. Biol. Chem. 271, 6389 (1996)], Before irradiation, cells were sometimes incubated with the tyrosine kinase inhibitor genistein (30 μg/ml for 1 hour) at 37°C to examine the role of PTK in radiation-induced apoptosis as described (2) [F. M. Uckun et al., ibid. 90, 252 (1993)].
37. 137Cs irradiator; J. L. Shephard, Glendale, CA, Model Mark I) with 4 or 8 Gy γ-irradiation at a dose rate of 1 Gy/min under aerobic conditions as described [F. M. Uckun et al., Cancer Res. 53, 1431 (1993); F. M. Uckun et al., J Clin. Invest. 91, 1044 (1993); F. M. Uckun et al., J. Biol. Chem. 271, 6389 (1996)], Before irradiation, cells were sometimes incubated with the tyrosine kinase inhibitor genistein (30 μg/ml for 1 hour) at 37°C to examine the role of PTK in radiation-induced apoptosis as described (2) [F. M. Uckun et al., ibid. 90, 252 (1993)].
38. 137Cs irradiator; J. L. Shephard, Glendale, CA, Model Mark I) with 4 or 8 Gy γ-irradiation at a dose rate of 1 Gy/min under aerobic conditions as described [F. M. Uckun et al., Cancer Res. 53, 1431 (1993); F. M. Uckun et al., J Clin. Invest. 91, 1044 (1993); F. M. Uckun et al., J. Biol. Chem. 271, 6389 (1996)], Before irradiation, cells were sometimes incubated with the tyrosine kinase inhibitor genistein (30 μg/ml for 1 hour) at 37°C to examine the role of PTK in radiation-induced apoptosis as described (2) [F. M. Uckun et al., ibid. 90, 252 (1993)].
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40. 6 cells were suspended in MC540 (5 μg/ml) and PI (10 μg/ml) and kept in the dark at 4°C, Whole cells were analyzed with a FACStar Plus flow cytometer (Becton Dickinson). All analyses were done at 488-nm excitation from an argon laser. MC540 and PI emissions were split with a 600-nm short-pass dichroic mirror; a 575-nm band pass filter was placed in front of one photomultiplier tube to measure MC540 emission, and a 635-nm band pass filter was used for PI emission. When DT-40 cells were exposed to 4-Gy γ-rays, the percentage of apoptotic cells, as determined by dual MC540/PI fluorescence, increased within 8 hours after radiation from 7 to 65%. By comparison, only 17% of DT-40 cells preincubated with genistein (30 μg/ml) for 1 hour at 37°C before radiation exposure were apoptotic at 8 hours after radiation.
41. 6 cells were suspended in MC540 (5 μg/ml) and PI (10 μg/ml) and kept in the dark at 4°C, Whole cells were analyzed with a FACStar Plus flow cytometer (Becton Dickinson). All analyses were done at 488-nm excitation from an argon laser. MC540 and PI emissions were split with a 600-nm short-pass dichroic mirror; a 575-nm band pass filter was placed in front of one photomultiplier tube to measure MC540 emission, and a 635-nm band pass filter was used for PI emission. When DT-40 cells were exposed to 4-Gy γ-rays, the percentage of apoptotic cells, as determined by dual MC540/PI fluorescence, increased within 8 hours after radiation from 7 to 65%. By comparison, only 17% of DT-40 cells preincubated with genistein (30 μg/ml) for 1 hour at 37°C before radiation exposure were apoptotic at 8 hours after radiation.
42. 6 cells were suspended in MC540 (5 μg/ml) and PI (10 μg/ml) and kept in the dark at 4°C, Whole cells were analyzed with a FACStar Plus flow cytometer (Becton Dickinson). All analyses were done at 488-nm excitation from an argon laser. MC540 and PI emissions were split with a 600-nm short-pass dichroic mirror; a 575-nm band pass filter was placed in front of one photomultiplier tube to measure MC540 emission, and a 635-nm band pass filter was used for PI emission. When DT-40 cells were exposed to 4-Gy γ-rays, the percentage of apoptotic cells, as determined by dual MC540/PI fluorescence, increased within 8 hours after radiation from 7 to 65%. By comparison, only 17% of DT-40 cells preincubated with genistein (30 μg/ml) for 1 hour at 37°C before radiation exposure were apoptotic at 8 hours after radiation.
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50. Antibodies directed against BTK (19), SYK [R. B. Rowley, A. L Burkhardt, H.-G. Chao, G. R. Matsueda, J. B. Bolen, J. Bol. Chem. 270, 11590 (1995)], and LYN (4) have been described previously. Polyclonal antibodies to BTK were generated by immunization of rabbits with glutathione-S-transferase fusion proteins (Pharmacia LKB Biotechnology) containing the first 150 amino acids of BTK. Immunoprecipitations, immune-complex protein kinase assays, and immunoblotting were done as described [F. M. Uckun et al., J. Biol. Chem. 268, 21172 (1993); F. M. Uckun et al., Science 267. 886 (1995)].
51. Antibodies directed against BTK (19), SYK [R. B. Rowley, A. L Burkhardt, H.-G. Chao, G. R. Matsueda, J. B. Bolen, J. Bol. Chem. 270, 11590 (1995)], and LYN (4) have been described previously. Polyclonal antibodies to BTK were generated by immunization of rabbits with glutathione-S-transferase fusion proteins (Pharmacia LKB Biotechnology) containing the first 150 amino acids of BTK. Immunoprecipitations, immune-complex protein kinase assays, and immunoblotting were done as described [F. M. Uckun et al., J. Biol. Chem. 268, 21172 (1993); F. M. Uckun et al., Science 267. 886 (1995)].
52. Antibodies directed against BTK (19), SYK [R. B. Rowley, A. L Burkhardt, H.-G. Chao, G. R. Matsueda, J. B. Bolen, J. Bol. Chem. 270, 11590 (1995)], and LYN (4) have been described previously. Polyclonal antibodies to BTK were generated by immunization of rabbits with glutathione-S-transferase fusion proteins (Pharmacia LKB Biotechnology) containing the first 150 amino acids of BTK. Immunoprecipitations, immune-complex protein kinase assays, and immunoblotting were done as described [F. M. Uckun et al., J. Biol. Chem. 268, 21172 (1993); F. M. Uckun et al., Science 267. 886 (1995)].
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83. M. Takata and T. Kurosaki, unpublished observations.
84. Supported by National Institutes of Health grants R01-CA-42111 and R01-CA-42633. F.M.U. is a Stohlman Scholar of the Leukemia Society of America.