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PubMed2004

Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication: evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication.

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.

Antibodies, Monoclonal↗
PubMed2005

Identification and functional evaluation of cellular and viral factors involved in the alteration of nuclear architecture during herpes simplex virus 1 infection.

Herpes simplex virus 1 (HSV-1) replicates in the nucleus of host cells and radically alters nuclear architecture as part of its replication process. Replication compartments (RCs) form, and host chromatin is marginalized. Chromatin is later dispersed, and RCs spread past it to reach the nuclear edge. Using a lamin A-green fluorescent protein fusion, we provide direct evidence that the nuclear lamina is disrupted during HSV-1 infection and that the UL31 and UL34 proteins are required for this. We show nuclear expansion from 8 h to 24 h postinfection and place chromatin rearrangement and disruption of the lamina in the context of this global change in nuclear architecture. We show HSV-1-induced disruption of the localization of Cdc14B, a cellular protein and component of a putative nucleoskeleton. We also show that UL31 and UL34 are required for nuclear expansion. Studies with inhibitors of globular actin (G-actin) indicate that G-actin plays an essential role in nuclear expansion and chromatin dispersal but not in lamina alterations induced by HSV-1 infection. From analyses of HSV infections under various conditions, we conclude that nuclear expansion and chromatin dispersal are dispensable for optimal replication, while lamina rearrangement is associated with efficient replication.

Actins↗
PubMed2001

Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures.

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.

Antigens, Viral↗
PubMed2001

Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9.

The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathione S-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

Amino Acid Sequence↗
PubMed1991

Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein.

We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein.

Animals↗
PubMed2001

Fluorescent tagging of herpes simplex virus tegument protein VP13/14 in virus infection.

The cellular site of herpesvirus tegument assembly has yet to be defined. We have previously used a recombinant herpes simplex virus type 1 expressing a green fluorescent protein (GFP)-tagged tegument protein, namely VP22, to show that VP22 is localized exclusively to the cytoplasm during infection. Here we have constructed a similar virus expressing another fluorescent tegument protein, YFP-VP13/14, and have visualized the intracellular localization of this second tegument protein in live infected cells. In contrast to VP22, VP13/14 is targeted predominantly to the nuclei of infected cells at both early and late times in infection. More specifically, YFP-13/14 localizes initially to the nuclear replication compartments and then progresses into intense punctate domains that appear at around 12 h postinfection. At even later times this intranuclear punctate fluorescence is gradually replaced by perinuclear micropunctate and membranous fluorescence. While the vast majority of YFP-13/14 seems to be targeted to the nucleus, a minor subpopulation also appears in a vesicular pattern in the cytoplasm that closely resembles the pattern previously observed for GFP-22. Moreover, at late times weak fluorescence appears at the cell periphery and in extracellular virus particles, confirming that YFP-13/14 is assembled into virions. This predominantly nuclear targeting of YFP-13/14 together with the cytoplasmic targeting of VP22 may imply that there are multiple sites of tegument protein incorporation along the virus maturation pathway. Thus, our YFP-13/14-expressing virus has revealed the complexity of the intracellular targeting of VP13/14 and provides a novel insight into the mechanism of tegument, and hence virus, assembly.

Animals↗
PubMed1996

Intracellular localization of the herpes simplex virus type-1 origin binding protein, UL9.

UL9 is the origin binding protein of herpes simplex virus type-1 (HSV-1). A UL9-specific monoclonal antibody (17B) whose epitope maps to the N-terminal 33 amino acids was used to study the localization of UL9 in infected and transfected cells. We demonstrate the colocalization of UL9 and the HSV-1 single-strand DNA binding protein (ICP8 or UL29) in replication compartments, sites of viral DNA synthesis. On the other hand, UL9 does not completely colocalize with ICP8 in prereplicative sites, structures observed under conditions that inhibit viral DNA polymerase. Cells transfected with various deletion or pyruvate kinase fusion constructs were analyzed by indirect immunofluorescence assay to define the nuclear localization signal (NLS) of UL9. Deletion analysis showed that the region required for nuclear localization lies within the C-terminal DNA binding domian (amino acids 535-851). Various regions of UL9 were tested in fusion constructs for their ability to direct the normally cytoplasmic chicken pyruvate kinase protein to the nucleus. A fusion construct containing the carboxy-terminal 107 residues (amino acids 745-851) localized efficiently to the nucleus, whereas a fusion construct containing the N-terminal 660 amino acids of UL9 was unable to do so. Mutations designed to alter a potential NLS sequence (793-KREFAGARFKLR-804) within the C-terminal 107 residues result in a mutant UL9 protein which falls to localize efficiently to the nucleus. These results suggest that the major NLS of UL9 maps within the C-terminal 107 amino acids.

Animals↗
PubMed2004

Differential virus evolution in blood and genital tract of HIV-infected females: evidence for the involvement of drug and non-drug resistance-associated mutations.

There is increasing evidence that male or female genital tract represent a distinct replication compartment for human immunodeficiency virus type 1 (HIV-1) and that such compartments may serve as a virus reservoir. Forty-four paired plasma and vaginal samples from HIV-infected females undergoing HAART were collected to examine the viral responses to antiretroviral therapy and to assess the possible role of the vaginal tract as a reservoir for drug-resistant variants. Twenty-one females had detectable viral RNA both in plasma and vaginal fluid, whereas 14 females had detectable virus only in plasma. Twelve paired samples were used to analyze HIV-1 pol sequences for the presence of drug resistance-associated mutations. Nine of the twelve paired samples exhibited discordant drug resistance mutation patterns. The other three females showed identical drug resistance-associated mutations. However, further examination of protease and RT showed numerous non-drug-associated mutations that corresponded to predefined CTL epitopes. These non-drug-associated mutations were different between plasma and vaginal viruses, suggesting that evolution of HIV-1 was independent in these two compartments.

Adult↗
PubMed2025

Spatial Mapping and Interactome Profiling of m6A-Modified R-Loops via Chemically Inducible Split-APEX2 Proximity Labeling.

m6A-Modified R-loops (m6A-R-loops) play crucial roles in epigenetic regulation and genome stability, yet resolving their spatial distribution and protein interactomes in live cells remains challenging. To address this, we developed m6A-R-loop proximity labeling (m6A-RLPL), a chemically inducible split-APEX2 proximity labeling technology integrating dual-target recognition using the RNA-DNA hybrid binding domain of RNase H1 for R-loop targeting and m6A reader protein's YTH domain for m6A recognition, coupled with an abscisic acid (ABA)-inducible dimerization system for signal amplification. This technology revealed host m6A-R-loops enriched with nucleoli under normal conditions. When applied to herpes simplex virus (HSV) infection, it further demonstrated viral m6A-R-loops undergoing dramatic accumulation within phase-separated granules in replication compartments during late-stage infection. Proximity proteomics identified ZC3H4 and CCDC124 as essential regulators maintaining these structures, which serve as transcription sites for HSV late genes, with disruption selectively impairing viral transcription. m6A-RLPL establishes a generalizable approach for spatially resolved profiling of m6A-R-loop interactomes and organizational dynamics in living systems.

Humans↗
PubMed2000

A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication.

As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment.

Agrobacterium tumefaciens↗
PubMed2006

Epstein-Barr virus protein kinase BGLF4 is a virion tegument protein that dissociates from virions in a phosphorylation-dependent process and phosphorylates the viral immediate-early protein BZLF1.

Epstein-Barr virus (EBV) BGLF4 is a viral protein kinase that is expressed in the lytic phase of infection and is packaged in virions. We report here that BGLF4 is a tegument protein that dissociates from the virion in a phosphorylation-dependent process. We also present evidence that BGLF4 interacts with and phosphorylates BZLF1, a key viral regulator of lytic infection. These conclusions are based on the following observations. (i) In in vitro tegument release assays, a significant fraction of BGLF4 was released from virions in the presence of physiological NaCl concentrations. (ii) Addition of physiological concentrations of ATP and MgCl(2) to virions enhanced BGLF4 release, but phosphatase treatment of virions significantly reduced BGLF4 release. (iii) A recombinant protein containing a domain of BZLF1 was specifically phosphorylated by purified recombinant BGLF4 in vitro, and BGLF4 altered BZLF1 posttranslational modification in vivo. (iv) BZLF1 was specifically coimmunoprecipitated with BGLF4 in 12-O-tetradecanoylphorbol-13-acetate-treated B95-8 cells and in COS-1 cells transiently expressing both of these viral proteins. (v) BGLF4 and BZLF1 were colocalized in intranuclear globular structures, resembling the viral replication compartment, in Akata cells treated with anti-human immunoglobulin G. Our results suggest that BGLF4 functions not only in lytically infected cells by phosphorylating viral and cellular targets but also immediately after viral penetration like other herpesvirus tegument proteins.

Cell Line↗
PubMed1999

Modified VP22 localizes to the cell nucleus during synchronized herpes simplex virus type 1 infection.

The UL49 gene product (VP22) of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is a virion phosphoprotein which accumulates inside infected cells at late stages of infection. We previously (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994) discovered that the form of VP22 packaged into infectious virions differed from VP22 extracted from infected-cell nuclei in that the virion-associated form had a higher electrophoretic mobility in denaturing gels. Based on these results, we proposed that VP22 in virions was "undermodified" in some way. The goal of this study is to document the biological and biochemical properties of VP22 throughout the entire course of a productive HSV-1 infection. We now report the following. (i) VP22 found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear, as measured by indirect immunofluorescence. (ii) Using a synchronized infection system, we determined that VP22 exists predominantly in the cytoplasm early in infection and accumulates in the nucleus late in infection. (iii) While cytoplasmic VP22 colocalizes with the HSV-1 glycoprotein D early in infection, the nuclear form of VP22 is not restricted to replication compartments which accumulate ICP4. (iv) VP22 migrates as at least three unique electrophoretic species in denaturing sodium dodecyl sulfate-DATD-polyacrylamide gels. VP22a, VP22b, and VP22c have high, intermediate, and low mobility, respectively. (v) The relative distribution of the various forms of VP22 derived from infected whole-cell extracts varies during the course of infection such that low-mobility species predominate at early times and high-mobility forms accumulate later. (vi) The highest-mobility forms of VP22 partition with the cytoplasmic fraction of infected cells, while the lowest-mobility forms are associated with the nuclear fraction. (vii) Finally, full-length VP22 which partitions in the nucleus incorporates radiolabel from [32P]orthophosphate whereas cytoplasmic VP22 does not. Based on these results, we conclude that modification of VP22 coincides with its appearance in the nucleus during the course of productive HSV-1 infection.

Animals↗
PubMed2000

Analysis of HCF, the cellular cofactor of VP16, in herpes simplex virus-infected cells.

Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperature-sensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.

Animals↗
PubMed2001

U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids.

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U(L)31 protein fused to glutathione-S-transferase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demonstrated to specifically recognize the U(L)31 and U(L)34 proteins of approximately 34,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U(L)34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U(L)31 null virus. When transiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U(S)3-encoded protein kinase, previously shown to phosphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 kinase is required for even distribution of U(L)31 and U(L)34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U(L)31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)31 and U(L)34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.

Animals↗
PubMed2002

Sequential localization of two herpes simplex virus tegument proteins to punctate nuclear dots adjacent to ICP0 domains.

The subcellular localization of herpes simplex virus tegument proteins during infection is varied and complex. By using viruses expressing tegument proteins tagged with fluorescent proteins, we previously demonstrated that the major tegument protein VP22 exhibits a cytoplasmic localization, whereas the major tegument protein VP13/14 localizes to nuclear replication compartments and punctate domains. Here, we demonstrate the presence of a second minor population of VP22 in nuclear dots similar in appearance to those formed by VP13/14. We have constructed the first-described doubly fluorescence-tagged virus expressing VP22 and VP13/14 as fusion proteins with cyan fluorescent protein and yellow fluorescent protein, respectively. Visualization of both proteins within the same live infected cells has indicated that these two tegument proteins localize to the same nuclear dots but that VP22 appears there earlier than VP13/14. Further studies have shown that these tegument-specific dots are detectable as phase-dense bodies as early as 2 h after infection and that they are different from the previously described nuclear domains that contain capsid proteins. They are also different from the ICP0 domains formed at cellular nuclear domain 10 sites early in infection but, in almost all cases, are located in juxtaposition to these ICP0 domains. Hence, these tegument proteins join a growing number of proteins that are targeted to discrete nuclear domains in the herpesvirus-infected cell nucleus.

Animals↗
PubMed2000

Expression of the human mismatch repair gene hMSH2: a potential marker for urothelial malignancy.

BACKGROUND: The human mismatch repair (MMR) gene hMSH2 (human mutS homolog-2) is a DNA repair gene that has been reported to be mutated in 40% of hereditary nonpolyposis colon cancer (HNPCC) kindreds and a small percentage of sporadic tumors. HNPCC is a cancer predisposition syndrome with an increased risk of carcinoma of the colon, endometrium, stomach, small intestine, ovary, ureter, and renal pelvis. Immunohistochemical analysis demonstrated increased hMSH2 expression in sporadic colon carcinoma and in the replicative compartment of normal epithelium. A recent immunohistochemical analysis of hMSH2 in bladder tumors correlated reduced hMSH2 expression with recurrence and higher tumor grade. In the current study, we examined hMSH2 expression in urothelial malignancy using immunohistochemical analysis and developed a molecular assay for the detection of hMSH2 expression in bladder washes. METHODS: Immunohistochemical analysis of 17 tumors from the genitourinary tract and reverse transcription coupled with polymerase chain reaction (RT-PCR) of 40 bladder washes were used to investigate hMSH2 expression in noninvasive and invasive urothelial malignancies. RESULTS: Increased expression of hMSH2 was detected in all tumors examined using immunohistochemical analysis independent of grade or stage. Reverse transcription-PCR of hMSH2 mRNA from bladder washes detected 17 of 21 patients with primary or recurrent urothelial neoplasms or tumors involving the urothelial system. Four patients with urothelial malignancies without detectable hMSH2 expression from their bladder washes had high grade lesions. Ten of 13 patients without pathologic or cystoscopic evidence of bladder tumors were negative for hMSH2 expression in bladder washes. Two patients with bladder tumors and bladder washes that were positive for hMSH2 subsequently were found to be negative for hMSH2 after treatment of their tumors and at last follow-up had remained recurrence free for at least 1 year. CONCLUSIONS: The results of the current study suggest that hMSH2 expression is increased in low and high grade urothelial neoplasms, similar to the expression pattern in sporadic colon carcinoma. However, a fraction of high grade lesions may not express hMSH2 as detected by RT-PCR from bladder washes. The ability to detect hMSH2 expression in bladder washes may allow the use of hMSH2 expression as a marker for urothelial malignancy. In addition, the ability to define hMSH2 deficient tumors using bladder washes may have prognostic significance in the treatment of patients with urothelial carcinoma.

Adenosine Triphosphatases↗
PubMed1996

Expression of the human mismatch repair gene hMSH2 in normal and neoplastic tissues.

Hereditary nonpolyposis colorectal cancer is caused by inherited mutations of mismatch repair genes. We developed monoclonal antibodies to the prototype human mismatch repair gene hMSH2 and used them to detect an immunoreactive protein of M(r) 100,000 in mismatch-proficient cell lines. In addition, a M(r) 150,000 protein coimmunoprecipitated with the hMSH2 gene product in cell lines expressing hMSH2. Immunohistochemistry demonstrated that the hMSH2 protein was exclusively nuclear. Whereas the hMSH2 protein was expressed in a variety of tissues, the most striking pattern was observed in esophageal and intestinal epithelia, where expression was limited to the replicating compartment. Neoplastic cells within benign and malignant mismatch repair-proficient tumors expressed the protein, but no hMSH2 immunoreactivity was observed in the colorectal tumors of patients with germline hMSH2 mutation. These results have implications for tumorigenic mechanisms and, potentially, for diagnosis.

Animals↗
PubMed1998

Increased cell proliferation characterizes Crohn's disease.

Patients with long-standing Crohn's disease (CD), a chronic inflammatory intestinal disease, are at increased risk for intestinal cancer. The neoplasia likely results, in part, from deregulated cell proliferation, which allows mutations to become fixed in the crypt progenitor cells. We postulated that tissues derived from patients with CD would exhibit increased mucosal proliferation. Therefore, we examined specimens from 27 consecutive patients with chronic CD with a monoclonal antibody directed against the proliferation marker, Ki-67. The tissues were evaluated histologically, and the Ki-67 immunostaining patterns were recorded. The antibody to Ki-67 stained the bases of the crypts in both the small and large intestines. The mean number of Ki-67 immunoreactive cells in the normal crypt was 34.1 versus 95.1 in the regenerative mucosa and O in areas of pyloric metaplasia (P < .00001). Ki-67 staining of the mucosa of patients with CD confirmed that cell proliferation is markedly increased and that the replicating compartment of each crypt during regeneration is expanded. We concluded that the increased cell proliferation might predispose the mucosa to mutational events, thereby increasing the cancer risk in these patients. The lack of proliferation in areas of pyloric metaplasia might represent a mucosal adaptive response of the lower crypt that decreases the number of cycling cells vulnerable to genetic damage. Furthermore, growth factors produced by these cells might promote healing of the damaged mucosa.

Adult↗