TY - JOUR
T1 - The human retinoblastoma gene product suppresses ceramide-induced apoptosis in human bladder tumor cells
AU - McConkey, David J.
AU - Goodrich, David
AU - Bucana, Cora
AU - Klostergaard, Jim
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1996
Y1 - 1996
N2 - The retinoblastoma gene product, Rb, has previously been implicated as an obligatory component in the antiproliferative effects mediated by the lipid second messenger, ceramide. We have evaluated both the apoptotic effects and the effects on cell cycle distribution of the exogenous cell-permeable ceramide, N-hexanoyl-D-sphingosine, in an Rb-null human bladder tumor cell line, 5637, as well as in retrovirally infected, Rb(+) clones derived there from. These cell lines demonstrated comparable sensitivity to N-hexanoyl-D-sphingosine in a neutral red dye uptake assay. Exposure of the Rb-null parental cell line to 20 μM N-hexanoyl-D-sphingosine for 24 h resulted in a classical pattern of DNA fragmentation that was accompanied by apoptotic nuclear morphological alterations. In contrast, the Rb(+) clones demonstrated suppression of DNA fragmentation in response to N-hexanoyl-D-sphingosine. Similarly, the frequency and degree of alteration of nuclear morphology in Rb(+) cells was also suppressed. Flow cytometric analysis of the parental and infected clones indicated that expression of Rb was without effect on their cell cycle distribution, with or without exposure to N-hexanoyl-D-sphingosine for 25 h; tunel assay confirmed that in this time frame apoptotic cells were far less frequent in the Rb(+) clones than in the parental 5637 cells. Human tumor cell lines derived from three other histological origins, breast and prostatic carcinomas and osteogenic sarcoma, also demonstrated very similar cytotoxic sensitivities to N-hexanoyl-D-sphingosine, irrespective of the expression of Rb. We conclude that Rb is not required for ceramide-induced apoptosis and that Rb can actually inhibit the DNA fragmentation and nuclear morphological changes associated with classical apoptosis.
AB - The retinoblastoma gene product, Rb, has previously been implicated as an obligatory component in the antiproliferative effects mediated by the lipid second messenger, ceramide. We have evaluated both the apoptotic effects and the effects on cell cycle distribution of the exogenous cell-permeable ceramide, N-hexanoyl-D-sphingosine, in an Rb-null human bladder tumor cell line, 5637, as well as in retrovirally infected, Rb(+) clones derived there from. These cell lines demonstrated comparable sensitivity to N-hexanoyl-D-sphingosine in a neutral red dye uptake assay. Exposure of the Rb-null parental cell line to 20 μM N-hexanoyl-D-sphingosine for 24 h resulted in a classical pattern of DNA fragmentation that was accompanied by apoptotic nuclear morphological alterations. In contrast, the Rb(+) clones demonstrated suppression of DNA fragmentation in response to N-hexanoyl-D-sphingosine. Similarly, the frequency and degree of alteration of nuclear morphology in Rb(+) cells was also suppressed. Flow cytometric analysis of the parental and infected clones indicated that expression of Rb was without effect on their cell cycle distribution, with or without exposure to N-hexanoyl-D-sphingosine for 25 h; tunel assay confirmed that in this time frame apoptotic cells were far less frequent in the Rb(+) clones than in the parental 5637 cells. Human tumor cell lines derived from three other histological origins, breast and prostatic carcinomas and osteogenic sarcoma, also demonstrated very similar cytotoxic sensitivities to N-hexanoyl-D-sphingosine, irrespective of the expression of Rb. We conclude that Rb is not required for ceramide-induced apoptosis and that Rb can actually inhibit the DNA fragmentation and nuclear morphological changes associated with classical apoptosis.
KW - Bladder carcinoma
KW - DNA fragmentation
KW - N-hexanoyl-D-sphingosine
KW - Rb
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M3 - Article
C2 - 8895515
AN - SCOPUS:0029912856
SN - 0950-9232
VL - 13
SP - 1693
EP - 1700
JO - Oncogene
JF - Oncogene
IS - 8
ER -