Repeated BCG treatment of mouse bladder selectively stimulates small GTPases and HLA antigens and inhibits single-spanning uroplakins.
BACKGROUND
Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following repeated intravesical BCG therapy.


METHODS
Mice were transurethrally instilled with BCG or pyrogen-free on days 1, 7, 14, and 21. Seven days after the last instillation, urothelia along with the submucosa was removed and amplified ds-DNA was prepared from control- and BCG-treated bladder mucosa and used to generate suppression subtractive hybridization (SSH). Plasmids from control- and BCG-specific differentially expressed clones and confirmed by Virtual Northern were then purified and the inserts were sequenced and annotated. Finally, chromatin immune precipitation combined with real-time polymerase chain reaction assay (ChIP/Q-PCR) was used to validate SSH-selected transcripts.


RESULTS
Repeated intravesical BCG treatment induced an up regulation of genes associated with antigen presentation (B2M, HLA-A, HLA-DQA1, HLA-DQB2, HLA-E, HLA-G, IGHG, and IGH) and representatives of two IFNgamma-induced small GTPase families: the GBPs (GBP1, GBP2, and GBP5) and the p47GTPases (IIGTP1, IIGTP2, and TGTP). Genes expressed in saline-treated bladders but down-regulated by BCG included: the single-spanning uroplakins (UPK3a and UPK2), SPRR2G, GSTM5, and RSP 19.


CONCLUSIONS
Here we introduced a hypothesis-generator approach to determine key genes involved in the urothelium/sumbmucosa responses to BCG therapy. Urinary bladder responds to repeated BCG treatment by up-regulating not only antigen presentation-related genes, but also GBP and p47 small GTPases, both potentially serving to mount a resistance to the replication of the Mycobacterium. It will be of tremendous future interest to determine whether these immune response cascades play a role in the anti-cancer effects exerted by BCG.
Saban
Marcia R
MR
Department of Physiology, The University Oklahoma Health Sciences Center, Oklahoma City, USA. marcia-saban@ouhsc.edu
Hellmich
Helen L
HL
Simpson
Cindy
C
Davis
Carole A
CA
Lang
Mark L
ML
Ihnat
Michael A
MA
O'Donnell
Michael A
MA
Wu
Xue-Ru
XR
Saban
Ricardo
R
D000818:Animals; D017951:Antigen Presentation / Q000235:genetics* / Q000276:immunology; D001500:BCG Vaccine / Q000008:administration & dosage / Q000494:pharmacology*; D015152:Blotting, Northern; D047369:Chromatin Immunoprecipitation; D005260:Female; D020558:GTP Phosphohydrolases / Q000096:biosynthesis* / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000276:immunology*; D006183:H-2 Antigens / Q000096:biosynthesis / Q000235:genetics; D006680:HLA Antigens / Q000096:biosynthesis*; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis* / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013382:Subtraction Technique; D001743:Urinary Bladder / Q000187:drug effects* / Q000378:metabolism; D019459:Urothelium / Q000187:drug effects*
D001500:BCG Vaccine; D006183:H-2 Antigens; D006680:HLA Antigens; D000949:Histocompatibility Antigens Class II; D008565:Membrane Proteins; D012333:RNA, Messenger; D020558:GTP Phosphohydrolases