Abstract
BackgroundDespite 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.MethodsMice 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.ResultsRepeated 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 IFNγ-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.ConclusionHere 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.
Highlights
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
Virtual Northern Virtual Northern blot was performed in 14 differential clones obtained from the subtracted library enriched for control-specific sequences (Plate C-1: E8, A12, C7, B10, C11 and Plate C-2: A9, A12, B4, B6, B12, C12, E8, G8, H8) and 41 clones from the subtracted library enriched for BCG-specific sequences (Plate T-1: C1, D8, B10, E8, G7; Plate T-2: A11, B2, C10, D5, D11, F3, F5, G1; Plate T-3: A6, B4, B10, C9, D2, D6, D11, E1, E5, F5, G2, H3; Plate T-4: A2, B3, B9, C7, D4, D12, F10, G9, G11, H9; and Plate T-5: A3, B8, E9, E12, F11, H5)
Our results indicate that BCG induces both host adaptive immune response (HLA) and host mediated pathogen destruction (GTPases) with a down-regulation of uroplakins
Summary
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 unclear whether BCG is capable of altering gene expression beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. As an adjunct to transurethral resection, BCG is the treatment of choice for urothelial carcinoma in-situ (CIS) and is commonly used for recurrent or multi-focal Ta and high grade T1 bladder lesions [5,6]. It is not clear, how BCG alters the course of cystitis or cancer progression. One theory is that intravesical BCG corrects an aberrant immune imbalance in the bladder, leading to long-term symptomatic improvement [1]
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