BCG directly induces cell cycle arrest in human transitional carcinoma cell lines as a consequence of integrin cross-linking.
BACKGROUND
Current models of the mechanism by which intravesical BCG induces an anti-tumor effect in urothelial carcinoma propose a secondary cellular immune response as principally responsible. Our group has demonstrated that BCG mediated cross-linking of alpha51 integrin receptors present on the tumor surface elicits a complex biologic response involving AP1 and NF-kappaB signaling as well as the transactivation of immediate early genes. This study evaluated the direct biologic effect of cross-linking alpha5beta1 integrin on cell cycle progression and apoptosis in two human urothelial carcinoma cell lines.


METHODS
Two independent assays (MTT and Colony forming ability) were employed to measure the effect of alpha5beta1 cross-linking (antibody mediated or BCG) on cellular proliferation. Flow cytometry was employed to measure effect of BCG and alpha5beta1 antibody mediated cross-linking on cell cycle progression. Apoptosis was measured using assays for both DNA laddering and Caspase 3 activation.


RESULTS
Results demonstrate that integrin cross-linking by BCG, or antibody mediated crosslinking of alpha5beta1 resulted in a decrease in proliferating cell number. BCG treatment or alpha5beta1 cross-linking increased the percentage of cells in G0/G1, in both 253J and T24 cell lines. Peptide mediated blockade of integrin binding site using RGDS reversed the effect BCG on both proliferation and cell cycle arrest. Apoptosis in response to BCG was not identified by either DNA laddering or Caspase 3 activation.


CONCLUSIONS
These findings show that BCG exerts a direct cytostatic effect on human urothelial carcinoma cell lines. Cell cycle arrest at the G1/S interface is a mechanism by which BCG inhibits cellular proliferation. This effect is duplicated by antibody mediated cross-linking of alpha5beta1 and likely occurs as a consequence of crosslink-initiated signal transduction to cell cycle regulatory genes.
Chen
Fanghong
F
Department of Urology, Medical College of Wisconsin, Milwaukee, WI, USA. fanghongchen@hotmail.com
Zhang
Guangjian
G
Iwamoto
Yoshiki
Y
See
William A
WA
D000276:Adjuvants, Immunologic / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D001500:BCG Vaccine / Q000494:pharmacology* / Q000627:therapeutic use; D002295:Carcinoma, Transitional Cell / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D006801:Humans; D039081:Integrin alpha5beta1 / Q000187:drug effects* / Q000378:metabolism
D000276:Adjuvants, Immunologic; D001500:BCG Vaccine; D039081:Integrin alpha5beta1