Compound heterozygous MSH3 germline variants and associated tumor somatic DNA mismatch repair dysfunction.

We describe here an individual from a fourth family with germline compound heterozygous MSH3 germline variants and its observed biological consequences. The patient was initially diagnosed with invasive moderately-differentiated adenocarcinoma of the colon at the age of 43. Germline multigene panel testing revealed a pathogenic variant MSH3 c.2436-1 G > A and a variant of (initial) uncertain significance MSH3 c.3265 A > T (p.Lys1089*). Germline genetic testing of family members confirm the variants are in trans with the c.2436-1 G > A variant of paternal and the c.3265 A > T variant of maternal origin. Tumor DNA exhibits low levels of microsatellite instability and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). Tissue immunohistochemical staining for MSH3 demonstrated variant MSH3 protein is present in the cytoplasm and cell membrane but not in the nucleus of normal and tumor epithelial cells. Furthermore, variant MSH3 is accompanied by loss of nuclear MSH6 and a reduced level of nuclear MSH2 in some tumor cells, suggesting that the variant MSH3 protein may inhibit binding of MSH6 to MSH2.

The Human DNA Mismatch Repair Protein MSH3 Contains Nuclear Localization and Export Signals That Enable Nuclear-Cytosolic Shuttling in Response to Inflammation.

Inactivation of DNA mismatch repair propels colorectal cancer (CRC) tumorigenesis. CRCs exhibiting elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) show reduced nuclear MutS homolog 3 (MSH3) expression with surrounding inflammation and portend poor patient outcomes. MSH3 reversibly exits from the nucleus to the cytosol in response to the proinflammatory cytokine interleukin-6 (IL-6), suggesting that MSH3 may be a shuttling protein. In this study, we manipulated three putative nuclear localization (NLS1 to -3) and two potential nuclear export signals (NES1 and -2) within MSH3. We found that both NLS1 and NLS2 possess nuclear import function, with NLS1 responsible for nuclear localization within full-length MSH3. We also found that NES1 and NES2 work synergistically to maximize nuclear export, with both being required for IL-6-induced MSH3 export. We examined a 27-bp deletion (Δ27bp) within the polymorphic exon 1 that occurs frequently in human CRC cells and neighbors NLS1. With oxidative stress, MSH3 with this deletion (Δ27bp MSH3) localizes to the cytoplasm, suggesting that NLS1 function in Δ27bp MSH3 is compromised. Overall, MSH3's shuttling in response to inflammation enables accumulation in the cytoplasm; reduced nuclear MSH3 increases EMAST and DNA damage. We suggest that polymorphic sequences adjacent to NLS1 may enhance cytosolic retention, which has clinical implications for inflammation-associated neoplastic processes.

Inflammation-Associated Microsatellite Alterations Caused by MSH3 Dysfunction Are Prevalent in Ulcerative Colitis and Increase With Neoplastic Advancement.

OBJECTIVES: Inflammation-associated microsatellite alterations (also known as elevated microsatellite alterations at selected tetranucleotide repeats [EMAST]) result from IL-6-induced nuclear-to-cytosolic displacement of the DNA mismatch repair (MMR) protein MSH3, allowing frameshifts of dinucleotide or longer microsatellites within DNA. MSH3 also engages homologous recombination to repair double-strand breaks (DSBs), making MSH3 deficiency contributory to both EMAST and DSBs. EMAST is observed in cancers, but given its genesis by cytokines, it may be present in non-neoplastic inflammatory conditions. We examined ulcerative colitis (UC), a preneoplastic condition from prolonged inflammatory duration. METHODS: We assessed 70 UC colons without neoplasia, 5 UC specimens with dysplasia, 14 UC-derived colorectal cancers (CRCs), and 19 early-stage sporadic CRCs for microsatellite instability (MSI) via multiplexed polymerase chain reaction capable of simultaneous detection of MSI-H, MSI-L, and EMAST. We evaluated UC specimens for MSH3 expression via immunohistochemistry. RESULTS: UC, UC with dysplasia, and UC-derived CRCs demonstrated dinucleotide or longer microsatellite frameshifts, with UC showing coincident reduction of nuclear MSH3 expression. No UC specimen, with or without neoplasia, demonstrated mononucleotide frameshifts. EMAST frequency was higher in UC-derived CRCs than UC (71.4% vs 31.4%, P = 0.0045) and higher than early-stage sporadic CRCs (66.7% vs 26.3%, P = 0.0426). EMAST frequency was higher with UC duration >8 years compared with ≤8 years (40% vs 16%, P = 0.0459). DISCUSSION: Inflammation-associated microsatellite alterations/EMAST are prevalent in UC and signify genomic mutations in the absence of neoplasia. Duration of disease and advancement to neoplasia increases frequency of EMAST. MSH3 dysfunction is a potential contributory pathway toward neoplasia in UC that could be targeted by therapeutic intervention.

Inflammation-associated microsatellite alterations: Mechanisms and significance in the prognosis of patients with colorectal cancer.

Microsatellite alterations within genomic DNA frameshift as a result of defective DNA mismatch repair (MMR). About 15% of sporadic colorectal cancers (CRCs) manifest hypermethylation of the DNA MMR gene MLH1, resulting in mono- and di-nucleotide frameshifts to classify it as microsatellite instability-high (MSI-H) and hypermutated, and due to frameshifts at coding microsatellites generating neo-antigens, produce a robust protective immune response that can be enhanced with immune checkpoint blockade. More commonly, approximately 50% of sporadic non-MSI-H CRCs demonstrate frameshifts at di- and tetra-nucleotide microsatellites to classify it as MSI-low/elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) as a result of functional somatic inactivation of the DNA MMR protein MSH3 via a nuclear-to-cytosolic displacement. The trigger for MSH3 displacement appears to be inflammation and/or oxidative stress, and unlike MSI-H CRC patients, patients with MSI-L/EMAST CRCs show poor prognosis. These inflammatory-associated microsatellite alterations are a consequence of the local tumor microenvironment, and in theory, if the microenvironment is manipulated to lower inflammation, the microsatellite alterations and MSH3 dysfunction should be corrected. Here we describe the mechanisms and significance of inflammatory-associated microsatellite alterations, and propose three areas to deeply explore the consequences and prevention of inflammation's effect upon the DNA MMR system.

Production of truncated MBD4 protein by frameshift mutation in DNA mismatch repair-deficient cells enhances 5-fluorouracil sensitivity that is independent of hMLH1 status.

Methyl-CpG binding domain protein 4 (MBD4) is a DNA glycosylase that can remove 5-fluorodeoxyuracil from DNA as well as repair T:G or U:G mismatches. MBD4 is a target for frameshift mutation with DNA mismatch repair (MMR) deficiency, creating a truncated MBD4 protein (TruMBD4) that lacks its glycosylase domain. Here we show that TruMBD4 plays an important role for enhancing 5-fluorouracil (5FU) sensitivity in MMR-deficient colorectal cancer cells. We found biochemically that TruMBD4 binds to 5FU incorporated into DNA with higher affinity than MBD4. TruMBD4 reduced the 5FU affinity of the MMR recognition complexes that determined 5FU sensitivity by previous reports, suggesting other mechanisms might be operative to trigger cytotoxicity. To analyze overall 5FU sensitivity with TruMBD4, we established TruMBD4 overexpression in hMLH1-proficient or -deficient colorectal cancer cells followed by treatment with 5FU. 5FU-treated TruMBD4 cells demonstrated diminished growth characteristics compared to controls, independently of hMLH1 status. Flow cytometry revealed more 5FU-treated TruMBD4 cells in S phase than controls. We conclude that patients with MMR-deficient cancers, which show characteristic resistance to 5FU therapy, may be increased for 5FU sensitivity via secondary frameshift mutation of the base excision repair gene MBD4.

Efficacy of Adjuvant 5-Fluorouracil Therapy for Patients with EMAST-Positive Stage II/III Colorectal Cancer.

Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) is a genetic signature found in up to 60% of colorectal cancers (CRCs) that is caused by somatic dysfunction of the DNA mismatch repair (MMR) protein hMSH3. We have previously shown in vitro that recognition of 5-fluorouracil (5-FU) within DNA and subsequent cytotoxicity was most effective when both hMutSα (hMSH2-hMSH6 heterodimer) and hMutSß (hMSH2-hMSH3 heterodimer) MMR complexes were present, compared to hMutSα > hMutSß alone. We tested if patients with EMAST CRCs (hMutSß defective) had diminished response to adjuvant 5-FU chemotherapy, paralleling in vitro findings. We analyzed 230 patients with stage II/III sporadic colorectal cancers for which we had 5-FU treatment and survival data. Archival DNA was analyzed for EMAST (>2 of 5 markers mutated among UT5037, D8S321, D9S242, D20S82, D20S85 tetranucleotide loci). Kaplan-Meier survival curves were generated and multivariate analysis was used to determine contribution to risk. We identified 102 (44%) EMAST cancers. Ninety-four patients (41%) received adjuvant 5-FU chemotherapy, and median follow-up for all patients was 51 months. Patients with EMAST CRCs demonstrated improved survival with adjuvant 5FU to the same extent as patients with non-EMAST CRCs (P<0.05). We observed no difference in survival between patients with stage II/III EMAST and non-EMAST cancers (P = 0.36). There is improved survival for stage II/III CRC patients after adjuvant 5-FU-based chemotherapy regardless of EMAST status. The loss of contribution of hMSH3 for 5-FU cytotoxicity may not adversely affect patient outcome, contrasting patients whose tumors completely lack DNA MMR function (MSI-H).

EMAST is a Form of Microsatellite Instability That is Initiated by Inflammation and Modulates Colorectal Cancer Progression.

DNA mismatch repair (MMR) function is critical for correcting errors coincident with polymerase-driven DNA replication, and its proteins are frequent targets for inactivation (germline or somatic), generating a hypermutable tumor that drives cancer progression. The biomarker for defective DNA MMR is microsatellite instability-high (MSI-H), observed in ~15% of colorectal cancers, and defined by mono- and dinucleotide microsatellite frameshift mutations. MSI-H is highly correlated with loss of MMR protein expression, is commonly diploid, is often located in the right side of the colon, prognosticates good patient outcome, and predicts poor efficacy with 5-fluorouracil treatment. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is another form of MSI at tetranucleotide repeats that has been observed in multiple cancers, but its etiology and clinical relevance to patient care has only been recently illuminated. Specifically, EMAST is an acquired somatic defect observed in up to 60% of colorectal cancers and caused by unique dysfunction of the DNA MMR protein MSH3 (and its DNA MMR complex MutSß, a heterodimer of MSH2-MSH3), and in particular a loss-of-function phenotype due to a reversible shift from its normal nuclear location into the cytosol in response to oxidative stress and the pro-inflammatory cytokine interleukin-6. Tumor hypoxia may also be a contributor. Patients with EMAST colorectal cancers show diminished prognosis compared to patients without the presence of EMAST in their cancer. In addition to defective DNA MMR recognized by tetranucleotide (and di- and tri-nucleotide) frameshifts, loss of MSH3 also contributes to homologous recombination-mediated repair of DNA double stranded breaks, indicating the MSH3 dysfunction is a complex defect for cancer cells that generates not only EMAST but also may contribute to chromosomal instability and aneuploidy. Areas for future investigation for this most common DNA MMR defect among colorectal cancers include relationships between EMAST and chemotherapy response, patient outcome with aneuploid changes in colorectal cancers, target gene mutation analysis, and mechanisms related to inflammation-induced compartmentalization and inactivation for MSH3.

Interleukin 6 alters localization of hMSH3, leading to DNA mismatch repair defects in colorectal cancer cells.

BACKGROUND & AIMS: Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is the most common DNA mismatch repair defect in colorectal cancers, observed in approximately 60% of specimens. This acquired genotype correlates with metastasis and poor outcomes for patients, and is associated with intra-epithelial inflammation and heterogeneous nuclear levels of the mismatch repair protein hMSH3. Inflammation and accompanying oxidative stress can cause hMSH3 to change its intracellular location, but little is known about the source of oxidative stress in cancer cells. We investigated whether cytokines mediate this process. METHODS: We analyzed levels of interleukin 6 (IL6) and its receptor (IL6R) in human colon and lung cancer cell lines by flow cytometry and enzyme-linked immunosorbent assay; proteins were localized by immunofluorescence and immunoblot analyses. IL6 signaling was blocked with antibodies against IL6, soluble glycoprotein 130 Fc fragments, and the signal transducers and activators of transcription 3 inhibitor NSC74859; a constitutively active form of STAT3 was expressed in colon and lung cancer cell lines to replicate IL6R signaling. EMAST was detected by DNA fragment analysis. Immunohistochemistry was used to examine levels of IL6 in 20 colorectal tumor and adjacent nontumor tissues. RESULTS: Incubation of colon and lung cancer cell lines with IL6, but not other cytokines, caused hMSH3, but no other mismatch repair proteins, to move from the nucleus to the cytosol after generation of oxidative stress; inhibition of IL6 signaling prevented this shift. Expression of constitutively active STAT3 also caused hMSH3 to translocate from the nucleus to the cytoplasm in cancer cell lines. Incubation of cells with IL6 led to tetranucleotide frameshifts, the signature for EMAST. EMAST-positive colorectal tumors had significantly higher levels of IL6 than EMAST-negative tumors. CONCLUSIONS: IL6 signaling disrupts the nuclear localization of hMSH3 and DNA repair, leading to EMAST in cancer cell lines. Inflammatory cytokines might therefore promote genetic alterations in human cancer cells.

Acidic tumor microenvironment downregulates hMLH1 but does not diminish 5-fluorouracil chemosensitivity.

Human DNA mismatch repair (MMR) recognizes and binds 5-fluorouracil (5FU) incorporated into DNA and triggers a MMR-dependent cell death. Absence of MMR in a patient's colorectal tumor abrogates 5FU's beneficial effects on survival. Changes in the tumor microenvironment like low extracellular pH (pHe) may diminish DNA repair, increasing genomic instability. Here, we explored if low pHe modifies MMR recognition of 5FU, as 5FU can exist in ionized and non-ionized forms depending on pH. We demonstrate that MMR-proficient cells at low pHe show downregulation of hMLH1, whereas expression of TDG and MBD4, known DNA glycosylases for base excision repair (BER) that can remove 5FU from DNA, were unchanged. We show in vitro that 5FU within DNA pairs with adenine (A) at high and low pH (in absence of MMR and BER). Surprisingly, 5FdU:G was repaired to C:G in hMLH1-deficient cells cultured at both low and normal pHe, similar to MMR-proficient cells. Moreover, both hMSH6 and hMSH3, components of hMutSα and hMutSß, respectively, bound 5FU within DNA (hMSH6>hMSH3) but is influenced by hMLH1. We conclude that an acidic tumor microenvironment triggers downregulation of hMLH1, potentially removing the execution component of MMR for 5FU cytotoxicity, whereas other mechanisms remain stable to implement overall 5FU sensitivity.

Oxidative stress induces nuclear-to-cytosol shift of hMSH3, a potential mechanism for EMAST in colorectal cancer cells.

BACKGROUND: Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1's promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency. AIM: To examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency. METHODS: We measured -4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1⁻/⁻, hMSH6⁻/⁻, and hMSH3⁻/⁻ CRC cells. We observed the subcellular location of hMSH3 with oxidative stress. RESULTS: D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1⁻/⁻ and hMSH3⁻/⁻ cells, respectively, than in hMSH6⁻/⁻ or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10⁻4 mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10⁻4 mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H2O2 to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H2O2 treatment. CONCLUSION: EMAST is dependent upon the MMR background, with hMSH3⁻/⁻ more prone to frameshift mutations than hMSH6⁻/⁻, opposite to frameshift mutations observed for mononucleotide repeats. hMSH3⁻/⁻ mimics complete MMR failure (hMLH1⁻/⁻) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3's subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.

Both hMutSα and hMutSß DNA mismatch repair complexes participate in 5-fluorouracil cytotoxicity.

BACKGROUND: Patients with advanced microsatellite unstable colorectal cancers do not show a survival benefit from 5-fluorouracil (5-FU)-based chemotherapy. We and others have shown that the DNA mismatch repair (MMR) complex hMutSα binds 5-FU incorporated into DNA. Although hMutSß is known to interact with interstrand crosslinks (ICLs) induced by drugs such as cisplatin and psoralen, it has not been demonstrated to interact with 5-FU incorporated into DNA. Our aim was to examine if hMutSß plays a role in 5-FU recognition. METHODS: We compared the normalized growth of 5-FU treated cells containing either or both mismatch repair complexes using MTT and clonogenic assays. We utilized oligonucleotides containing 5-FU and purified baculovirus-synthesized hMutSα and hMutSß in electromobility shift assays (EMSA) and further analyzed binding using surface plasmon resonance. RESULTS: MTT and clonogenic assays after 5-FU treatment demonstrated the most cytotoxicity in cells with both hMutSα and hMutSß, intermediate cytotoxicity in cells with hMutSα alone, and the least cytotoxicity in cells with hMutSß alone, hMutSß binds 5-FU-modified DNA, but its relative binding is less than the binding of 5-FU-modified DNA by hMutSα. CONCLUSION: Cytotoxicity induced by 5-FU is dependent on intact DNA MMR, with relative cell death correlating directly with hMutSα and/or hMutSß 5-FU binding ability (hMutSα>hMutSß). The MMR complexes provide a hierarchical chemosensitivity for 5-FU cell death, and may have implications for treatment of patients with certain MMR-deficient tumors.

DNA mismatch repair proficiency executing 5-fluorouracil cytotoxicity in colorectal cancer cells.

BACKGROUND: 5-fluorouracil (5FU)-based chemotherapy is the standard treatment for advanced stage colorectal cancer (CRC) patients. Several groups including ours have reported that stage II-III colorectal cancer patients whose tumors retain DNA Mismatch repair (MMR) function derive a benefit from 5FU, but patients with tumors that lost MMR function do not. Although MMR recognition of 5FU incorporated in DNA has been demonstrated biochemically, it has not been demonstrated within cells to execute 5FU cytotoxicity. AIM: To establish an efficient construction model for 5FU within DNA and demonstrate that 5FU incorporated into DNA can trigger cellular cytotoxicity executed by the DNA MMR system. METHODS: We constructed a 5FdU-containing heteroduplex plasmid (5FdU plasmid) and 5FdU-containing linear dsDNA (5FdU linear DNA), and transfected these into MMR-proficient, hMLH1-/- and hMSH6-/- cells. We observed cell growth characteristics of both transfectants for 5FU-induced cytotoxicity. RESULTS: MMR- proficient cells transfected with the 5FdU plasmid but not the 5FdU linear DNA showed reduced cell proliferation by MTS and clonogenic assays, and demonstrated cell morphological change consistent with apoptosis. In MMR-deficient cells, neither the 5FdU plasmid nor 5FdU linear DNA induced cell growth or morphological changes different from controls. CONCLUSION: 5FdU as heteroduplex DNA in plasmid but not linear form triggered cytotoxicity in a MMR-dependent manner. Thus 5FU incorporated into DNA, separated from its effects on RNA, can be recognized by DNA MMR to trigger cell death.

Androgen ablation augments human HLA2.1-restricted T cell responses to PSA self-antigen in transgenic mice.

BACKGROUND: In recent years, there has been an increasing interest in targeting human prostate tumor-associated antigens (TAAs) for prostate cancer immunotherapy as an alternative to other therapeutic modalities. However, immunologic tolerance to TAA poses a significant obstacle to effective, TAA-targeted immunotherapy. We sought to investigate whether androgen deprivation would result in circumventing immune tolerance to prostate TAA by impacting CD8 cell responses. METHODS: To this end, we generated a transgenic mouse that expresses the human prostate-specific antigen (PSA) specifically in the prostate, and crossed it to the HLA-A2.1 transgenic mouse to evaluate how androgen deprivation affects human HLA A2.1-resticted T cell responses following immunization of PSA-expressing mice by vaccinia-PSA (PROSTVAC). RESULTS: Our PSA transgenic mouse showed restricted expression of PSA in the prostate and detectable circulating PSA levels. Additionally, PSA expression was androgen-dependent with reduced PSA expression in the prostate within 1 week of castration, and undetectable PSA by day 42 after castration as evaluated by ELISA. Castration of the PSA/A2.1 hybrid mouse prior to immunization with a PSA-expressing recombinant vaccinia virus resulted in a significant augmentation of PSA-specific cytotoxic lymphocytes. CONCLUSIONS: This humanized hybrid mouse model provides a well-defined system to gain additional insight into the mechanisms of immune tolerance to PSA and to test novel strategies aiming at circumventing immune tolerance to PSA and other TAA for targeted prostate cancer immunotherapy.

Loss of 15-hydroxyprostaglandin dehydrogenase expression contributes to bladder cancer progression.

Prostaglandin E2, which is known to contribute to cancer progression, is inactivated by the catabolic enzyme, 15-hydroxyprostaglandin dehydrogenase (PGDH), which has tumor-suppressor activity in lung, colon, breast, and gastric cancers. Therefore, we evaluated the expression of PGDH in human bladder cancer tissue specimens and cell lines. Immunoperoxidase staining of bladder cancer tissues demonstrated that (1) PGDH is highly expressed by normal urothelial cells but (2) reduced in many low stage (Ta/Tis) bladder cancers, and (3) PGDH is completely lost in most invasive bladder cancers. Of eight cancer cell lines tested, only two relatively well-differentiated bladder cancer cell lines, RT4 and UM-UC9, expressed PGDH. Moreover, inhibition of PGDH expression in well-differentiated RT4 cells using small inhibitory RNA or short hairpin RNA resulted in a more aggressive phenotype with increased motility and anchorage-independent growth. Additionally, PGDH knockdown affected prostaglandin E2 signaling as measured by cAMP generation. These data indicate that loss of PGDH expression contributes to a more malignant bladder cancer phenotype and may be necessary for bladder cancer development and/or progression.

A safety-modified SV40 Tag developed for human cancer immunotherapy.

Simian virus 40 (SV40)-like DNA sequences have been found in a variety of human tumors, raising the possibility that strategies targeting SV40 may provide a potential avenue for immunotherapy directed against SV40 large T Antigen (Tag)-expressing tumors. We generated a recombinant vaccinia (vac-mTag) expressing mTag and herein assessed the ability of mTag to transform cells and to interact with anti-oncoproteins, as well as screened for the presence of potential HLA-A2.1-restricted epitopes within mTag. We found that transfection of cells with mTag did not lead to their transformation. Also, we demonstrated that mTag protein is degraded rapidly in cells. In addition, our work revealed that mTag did not physically interact with certain anti-oncoproteins. Finally, two potential HLA-A2.1-restricted functional epitopes within mTag sequence were identified. Our results show that mTag lacks the oncogenicity of full-length Tag and harbors potential HLA-A2.1-restricted immunogenic epitopes, hence suggesting the safety of vac-mTag for use in cancer immunotherapy.

Interleukin-8 is essential for normal urothelial cell survival.

Interleukin-8 (IL-8; CXCL8) has been shown to play a role in multiple cellular processes. Here, we report an additional role of IL-8 as a growth and essential survival factor for normal human urothelial cells. Supplementing exogenous recombinant human IL-8 to normal urothelial cells promoted cell growth through the Akt pathway. Inhibition of IL-8 expression by small inhibitory RNA (siRNA) caused normal urothelial cells to die. Addition of recombinant human IL-8 rescued the normal urothelial cells treated with IL-8 siRNA. This rescue effect could be blocked by antibodies to the IL-8 receptor CXCR1 but not by CXCR2, suggesting that normal urothelial cells normally have IL-8 autocrine or paracrine activity for survival and growth mediated by CXCR1. IL-8 mRNA levels were lower in samples from patients with interstitial cystitis, a urinary bladder disorder associated with urothelial cell dysfunction and/or loss. Taken together, these results suggest that IL-8 is an important normal urothelial growth factor and is necessary for normal urothelial cell survival in vitro and in vivo. Lower IL-8 expression levels in the urinary bladder may contribute to pathophysiology of interstitial cystitis.

Loss of 15-hydroxyprostaglandin dehydrogenase expression disrupts urothelial differentiation.

OBJECTIVES: Urothelial differentiation is essential for the maintenance of urinary bladder function. We explored the expression and function of 15-hydroxyprostaglandin dehydrogenase (PGDH) during urothelial differentiation. METHODS: We evaluated expression of PGDH by Northern and Western blotting and immunostaining in human urothelial cultures, cell lines, and tissues. We determined enzymatic function using enzyme-linked immunosorbent assay. Small inhibitory ribonucleic acids were used to inhibit PGDH expression in human bladder cancer cells. RESULTS: We found PGDH messenger ribonucleic acid was increased in an in vitro model of human urothelial differentiation by Northern blotting. Western blotting of human bladder cancer cell lines showed expression in the well-differentiated RT4 cells and no expression in poorly differentiated UC3 cells. Immunostaining showed that PGDH expression increased with differentiation in normal bladder urothelium. The enzyme was functional in the well-differentiated RT4 human bladder cancer cell line. Inhibition of PGDH expression resulted in disruption of E-cadherin expression at cell-cell contacts in well-differentiated RT4 bladder cancer cells. CONCLUSIONS: These studies indicate that PGDH expression is associated with urothelial differentiation, and loss of PGDH expression results in disruption of urothelial differentiation.

Fas-mediated T cell deletion potentiates tumor antigen-specific tolerance in a mouse model of prostate cancer.

A pivotal obstacle to cancer immunotherapy is peripheral T cell tolerance to tumor-associated antigens (TAAs). Tolerance induction among mature T cells in the periphery operates through a variety of mechanisms, including anergy and apoptosis. Although Fas-FasL-mediated apoptosis is a well-defined tolerance inducing mechanism, direct evidence of its interference with TAA-specific immunity in vivo is still lacking. In this report, we used the TRAMP mouse, which expresses SV40 large T antigen (Tag) preferentially in the prostate and develops prostate tumors, as a model system to address the role of Fas-mediated apoptosis in regulating peripheral T cell tolerance. Using RT-PCR and tetramer staining to quantify TAA-specific TCR-expressing cytolytic T lymphocytes (CTLs), we have shown the presence of TAA-specific CTLs at higher levels in TRAMP mice than in syngeneic C57Bl/6 mice. Tag-specific immunization led to the expansion of Tag-specific CTLs in C57Bl/6 mice, and to their elimination in TRAMP mice. Interestingly, in TRAMP mice with deficient Fas (Hybrid TRAMP-lpr/lpr), Tag-specific CTL elimination in response to Tag immunization did not take place. The results of cytolytic-function assays were consistent with induction and elimination patterns of TAA-specific CTLs and those of RT-PCR and tetramer staining. In conclusion, our data show that Fas-mediated TAA-specific CTL apoptosis contributes to T cell tolerance and suggest that such tolerance could be potentiated following TAA-specific immunization.

RNA unwinding assay for DExD/H-box RNA helicases.

The evolutionarily conserved DExD/H-box proteins are essential for all RNA-related biological processes. They are thought to modulate the structure and function of specific RNAs and/or ribonucleoprotein particles by using their intrinsic RNA-dependent ATPase activities to achieve the desired conformational changes. A number of DExD/H-box proteins have been shown to unwind short RNA duplexes in vitro, a hallmark of the so-called RNA helicases or unwindases. However, some are unable to do so, perhaps because of requirements for cofactors. Here, we present a "solid-state" method that may allow investigators to overcome such problems.

Functional conservation of Dhh1p, a cytoplasmic DExD/H-box protein present in large complexes.

The DHH1 gene in the yeast Saccharomyces cerevisiae encodes a putative RNA helicase of remarkable sequence similarity to several other DExD/H-box proteins, including Xp54 in Xenopus laevis and Ste13p in Schizosaccharomyces pombe. We show here that over-expression of Xp54, an integral component of the stored messenger ribonucleoprotein (mRNP) particles, can rescue the loss of Dhh1p in yeast. Localization and sedimentation studies showed that Dhh1p exists predominantly in the cytoplasm and is present in large complexes whose sizes appear to vary according to the growth stage of the cell culture. In addition, deletion of dhh1, when placed in conjunction with the mutant dbp5 and ded1 alleles, resulted in a synergistically lethal effect, suggesting that Dhh1p may have a role in mRNA export and translation. Finally, similar to Ste13p, Dhh1p is required for sporulation in the budding yeast. Taken together, our data provide evidence that the functions of Dhh1p are conserved through evolution.