Involvement of non­triple helical type VI collagen α1 chain, NTH α1(VI), in the proliferation of cancer cells.

It has been reported that a polypeptide encoded by collagen type VI alpha 1 chain (COL6A1), one of the three α chains of type VI collagen, is strongly associated with the migration and invasion of highly metastatic human pancreatic cancer BxPC­M8 cells and excessive proliferation of LNCaP cells. We previously reported that non­triple helical type VI collagen α1 chain, NTH α1(VI), a non­triple helical polypeptide encoded by COL6A1, is not derived from type VI collagen and exists in cancer cell­conditioned media. Therefore, NTH α1(VI) may be involved in cancer cell migration, invasion, and proliferation. The active entity that promotes cellular behaviors in cancer remains unclear. Thus, we predicted that NTH α1(VI) has cancer­promoting activity, such as the ability to induce cell proliferation. This study was conducted to examine whether NTH α1(VI) and/or its derived peptides are involved in cancer cell proliferation. Highly metastatic human pancreatic S2­VP10 cells were used to explore the potential of COL6A1 knockdown in reducing cell proliferation. Moreover, S2­VP10 conditioned medium was assessed after molecular size­fractionation to determine whether the inhibitory effect of COL6A1 knockdown could be rescued by the medium. We showed that S2­VP10­conditioned medium contained COL6A1 polypeptide, but not COL6A2, suggesting that COL6A1 in the conditioned medium of S2­VP10 cells reflects the presence of NTH α1(VI). COL6A1 knockdown repressed S2­VP10 cell proliferation and this repression was rescued using the conditioned medium of S2­VP10 cells. The fraction of conditioned medium containing peptides smaller than 10 kDa rescued the inhibitory effect; however, the fraction containing polypeptides larger than 10 kDa, including NTH α1(VI), did not show rescue activity, indicating that NTH α1(VI) fragmentation is necessary for enhanced cancer cell proliferation. In conclusion, fragmentation of NTH α1(VI) into peptides <10 kDa is required for its cancer cell proliferation­promoting activity.

Identification of a common epitope in the sequences of COL4A1 and COL6A1 recognized by monoclonal antibody #141.

Identification of a type IV collagen α1 polypeptide in non-triple helical form [NTH α1(IV)], possibly involved in angiogenesis, introduces the further possibility of the existence of non-triple helical forms of other collagen chains. We previously reported that an anti-NTH α1(IV) monoclonal antibody #141 recognizes not only NTH α1(IV) but also a novel non-triple helical collagen polypeptide NTH α1(VI) encoded by COL6A1. In this study, we identified the recognition sequence in order to better understand the properties of antibody #141 and provide clues regarding the biological function of the two non-triple helical molecules. Additionally, we determined the common epitope between COL4A1 and COL6A1 as PXXGXPGLRG, with surface plasmon resonance analyses revealing KD values for the COL4A1 epitope as 5.56±1.81×10-9 M and for the COL6A1 epitope as 7.15±0.44×10-10 M. The specific recognition of NTH α1(IV) and NTH α1(VI) by antibody #141 can be explained by the common epitope sequence. Moreover, epitope localization supports previous finding that NTH α1(IV) and NTH α1(VI) differ in conformation from the α1 chains in triple-helical type IV and type VI collagen. These findings suggest that antibody #141 might be useful for diagnosis of type VI collagen myopathies.

Cell-based chemical fingerprinting identifies telomeres and lamin A as modifiers of DNA damage response in cancer cells.

Telomere maintenance by telomerase activity supports the infinite growth of cancer cells. MST-312, a synthetic telomerase inhibitor, gradually shortens telomeres at non-acute lethal doses and eventually induces senescence and apoptosis of telomerase-positive cancer cells. Here we report that MST-312 at higher doses works as a dual inhibitor of telomerase and DNA topoisomerase II and exhibits acute anti-proliferative effects on cancer cells and xenografted tumours in vivo. Our cell-based chemical fingerprinting approach revealed that cancer cells with shorter telomeres and lower expression of lamin A, a nuclear architectural protein, exhibited higher sensitivity to the acute deleterious effects of MST-312, accompanied by formation of telomere dysfunction-induced foci and DNA double-strand breaks. Telomere elongation and lamin A overexpression attenuated telomeric and non-telomeric DNA damage, respectively, and both conferred resistance to apoptosis induced by MST-312 and other DNA damaging anticancer agents. These observations suggest that sufficient pools of telomeres and a nuclear lamina component contribute to the cellular robustness against DNA damage induced by therapeutic treatment in human cancer cells.

Type VI collagen α1 chain polypeptide in non-triple helical form is an alternative gene product of COL6A1.

Expression of type IV collagen α1 chain in non-triple helical form, NTH α1(IV), is observed in cultured human cells, human placenta and rabbit tissues. Biological functions of NTH α1(IV) are most likely to be distinct from type IV collagen, since their biochemical characteristics are quite different. To explore the biological functions of NTH α1(IV), we prepared some anti-NTH α1(IV) antibodies. In the course of characterization of these antibodies, one antibody, #141, bound to a polypeptide of 140 kDa in size in addition to NTH α1(IV). In this study, we show evidence that the 140 kDa polypeptide is a novel non-triple helical polypeptide of type VI collagen α1 chain encoded by COL6A1, or NTH α1(VI). Expression of NTH α1(VI) is observed in supernatants of several human cancer cell lines, suggesting that the NTH α1(VI) might be involved in tumourigenesis. Reactivity with lectins indicates that sugar chains of NTH α1(VI) are different from those of the α1(VI) chain in triple helical form of type VI collagen, suggesting a synthetic mechanism and a mode of action of NTH α1(VI) is different from type VI collagen.

Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain.

This report describes the preparation and partial characterization of monoclonal antibodies that are reactive specifically with the nascently produced non-triple helical form of the type IV collagen α1 chain, designated as NTH α1(IV). These antibodies were nonreactive with the α1 chain of the type IV collagen in the triple-helical conformation. Three antibodies, #141, #179 and #370, with different epitopes in NTH α1(IV) were found to be reactive with the nascent polypeptide secreted from human normal cells and a human carcinoma cell line. The antibodies with different epitopes may provide a key method for elucidating the physiological function and tissue distribution of NTH α1(IV), which is distinct from the chain derived from triple-helical type IV collagen.

Non-Triple Helical Form of Type IV Collagen α1 Chain.

Type IV collagen with a triple-helical structure composed of three α chains is a major component of basement membrane. Previously, we reported that non-triple helical form of type IV collagen α1 chain (NTHα1(IV)) was isolated from human placenta and the culture media of human cells. In the present study, we report on the localization of NTH α1(IV) with a monoclonal antibody #370, exclusively reactive for the nascent chain, in the rabbit tissues. The staining was found on the basement membrane of blood vessels, of endomysium, of nerve, and of kidney but not on epithelial basement membrane. In a rabbit angiogenic model, #370 antibody staining was exclusively observed in neovascular tip region of endothelial cells, where no staining with anti-type IV collagen antibody was seen. Distinct localizations suggest that NTHα1(IV) is produced and stably deposited in endothelial cells and the surroundings under physiological conditions with some physiological roles in relation to the dynamics of vascular system.

Mast cell hyperplasia in the skin of Dsg4-deficient hypotrichosis mice, which are long-living mutants of lupus-prone mice.

Desmosomal cadherins are essential cell adhesion molecules expressed in the epidermis. We identified a mutation of a cadherin superfamily member, namely, desmoglein 4 (Dsg4), in early onset of death (EOD)( hage ) mice with hypotrichosis. The mutation was induced by the insertion of an early transposon II-beta into intron 8 of Dsg4. Mast cell hyperplasia was observed in the skin of EOD( hage ) mice. The abnormally expanded population of lpr T cells, i.e., CD4(-)CD8(-)B220(+)Thy1.2(+) alphabetaT cells, in the splenocytes of EOD mice was reduced in EOD( hage ) mice. Therefore, it was suspected that the long-living mutant EOD( hage ) mice were selected from lupus-prone EOD mice because of their immunological immaturity. These findings clearly indicate that Dsg4 is an important molecule for the formation of hair follicles and hypothesize that unorganized hyperplastic hair follicles in anagen due to the Dsg4 mutation provide niches for mast cell precursors in the skin.

Evaluation of the ameliorative effects of immunosuppressants on crescentic glomerulonephritis in SCG/Kj mice.

The therapeutic efficacy of immunosuppressants for treating rapidly progressive glomerulonephritis (RPGN) with crescent formation remains controversial. SCG/Kj mice spontaneously develop RPGN-like symptoms, characteristic of crescentic glomerulonephritis and systemic small vessel vasculitis, associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). We evaluated the "ameliorative", not prophylactic, effects of immunosuppressive agents, deoxyspergualin (DSG), cyclophosphamide (CYC) and prednisolone (PDN), on RPGN in these mice. DSG at intraperitoneal doses of 3 and 6 mg/kg, CYC at an oral dose of 12 mg/kg, or PDN at an intraperitoneal dose of 120 mg/kg was administered once a day for 21 days to female mice "at the onset of hematuria". A set of control SCG/Kj mice received only saline injections. DSG and CYC significantly prolonged survival, improved the proteinuria, hematuria and hyperuremia, and decreased the serum level of myeloperoxidase-ANCA. Moreover, DSG significantly suppressed the formation of crescents in glomeruli. PDN failed to affect any of the parameters. DSG might be useful for inducing remission in crescentic glomerulonephritis involved in RPGN.

A novel chemical compound, NK026680, targets dendritic cells to prolong recipient survival after rat liver grafting.

BACKGROUND: There is great interest in the recently developed immunosuppressant NK026680, which is a derivative of triazolopyrimidine. Its unique chemical structure and action mechanism are completely different from those of conventional immunosuppressants. METHODS: The present study was designed to investigate the effects of NK026680 on rat bone-marrow-derived dendritic cell (BMDC) differentiation and maturation in an in vitro culture system and its applicability in liver transplantation. RESULTS: NK026680 inhibited T-cell proliferation stimulated by alloantigen in a dose-dependent manner, but did not inhibit concanavalin A. The populations of OX6+CD161a cells and CD86+CD161a cells were suppressed in NK026680-treated dendritic cells (DCs). Exposure of DCs to NK026680 downregulated the interleukin (IL)-12 (p40, p35), interferon-gamma mRNA expression and upregulated IL-10, transforming growth factor-beta, in which impaired the ability of DC to stimulate T cell proliferation. Furthermore, oral administration of NK026680 for 14 days significantly prolonged liver allograft survival and limitation of T-cell responses and polarization toward a Th2 cytokine profile. CONCLUSIONS: These results demonstrate that NK026680 may have therapeutic potential for preventing allo-rejection in organ transplantation, acting at the step of immune response through inhibiting BMDC differentiation and maturation into potent antigen-presenting cells.

Trafficking of QD-conjugated MPO-ANCA in murine systemic vasculitis and glomerulonephritis model mice.

In systemic vasculitis, the serum level of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA) is significantly elevated with the progression of disease. We have established a model of murine systemic vasculitis by administration of MPO-ANCA and fungal mannoprotein to C57BL/6 mice. We examined the role of MPO and MPO-ANCA in the pathogenesis of glomerulonephritis and systemic vasculitis in this model using quantum dots (QDs). We demonstrated that QD-conjugated MPO-ANCA (ANCA-QD) visualized the translocation of MPO on the neutrophil membrane surface after stimulation with proinflammatory cytokines. We also observed that MPO translocation on neutrophils in both patients with rapid progressive glomerulonephritis and these model mice without any stimulation, suggesting that MPO translocation is certain to contribute to the development of glomerular lesion. In addition, blood flow on the kidney surface vessel was significantly decelerated in both SCG/Kj mice and this model, suggesting that ANCA induces the damage of blood vessel. These results indicate that MPO-ANCA and surface-translocated MPO on the activated neutrophils coordinately plays essential roles in the initial steps of the glomerulonephritis.

NK026680, a novel suppressant of dendritic cell function, prevents the development of rapidly progressive glomerulonephritis and perinuclear antineutrophil cytoplasmic antibody in SCG/Kj mice.

OBJECTIVE: NK026680 is a newly identified type of immunosuppressive agent that inhibits dendritic cell (DC) functions and consequently reduces the mortality of mice with experimental acute graft-versus-host disease. This study was undertaken to evaluate NK026680 suppression of DC functions in preventing development of rapidly progressive glomerulonephritis (RPGN) and perinuclear antineutrophil cytoplasmic antibodies (pANCA) in SCG/Kj mice. METHODS: Oral administration of NK026680 to SCG/Kj mice began when mice were 8-10 weeks old, before the onset of disease, and continued for 56 days. The efficacy of NK026680 was evaluated using the mortality of mice, the results of urinalysis, histopathologic evaluation for glomerular injury, and immunofluorescence staining for the detection of immune complex (IC) deposition in glomeruli, and by assessing lymphadenopathy and measuring autoantibody titers. RESULTS: Oral administration of NK026680 at a dosage of 25 mg/kg once daily or 50 mg/kg once daily significantly suppressed 1) spontaneous mortality, 2) proteinuria and hematuria, 3) blood urea nitrogen levels, 4) glomerular damage characterized histopathologically, 5) IC deposition in glomeruli, 6) the development of pANCA and anti-DNA antibodies, and 7) lymphadenopathy. CONCLUSION: The newly identified DC inhibitor, NK026680, prevented the onset of RPGN, autoantibody production, and lymphadenopathy in SCG/Kj mice, suggesting a crucial role for DC function in these autoimmune phenotypes. NK026680 may be a potent immunosuppressive agent for the treatment of ANCA-associated renovascular disorders.

Genetic dissection of vasculitis, myeloperoxidase-specific antineutrophil cytoplasmic autoantibody production, and related traits in spontaneous crescentic glomerulonephritis-forming/Kinjoh mice.

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse is a model of human crescentic glomerulonephritis and vasculitis associated with the production of the myeloperoxidase (MPO)-specific antineutrophil cytoplasmic autoantibody (MPO-ANCA). Although the disease is mediated initially by mutation of the Fas gene (lpr), SCG/Kj mice also have non-Fas predisposing genetic factors. To define these factors, genome-wide quantitative trait locus (QTL) mapping was performed on female (B(6)x SCG/Kj) F(2) intercross mice. Fourteen non-Fas QTLs were identified. QTLs of glomerulonephritis were located on chromosomes 1, 10, 13, 16, and 17, vasculitis on chromosomes 1 and 17, splenomegaly on chromosome 1, hypergammaglobulinemia on chromosomes 1, 2, 4, 6, 7, 11, 13, and 17, antinuclear Ab on chromosomes 1, 8, 10, and 12, and MPO-ANCA production on chromosomes 1 and 10. Significant QTLs derived from SCG/Kj on chromosomes 1, 2, 7, and 13 were designated Scg-1 to Scg-5, respectively, and those derived from B(6) on chromosomes 4, 6, 17, and 10 were designated Sxb-1 to Sxb-4, respectively. Two loci linked to MPO-ANCA production on chromosomes 1 and 10 were designated Man-1 and Man-2 (for MPO-ANCA), respectively. Although both Scg-1 and Scg-2 were on chromosome 1 and shared several functions, it was of interest that aberrant MPO-ANCA production was exclusively controlled by Man-1, the centromeric half region of the Scg-2 chromosomal segment. We also examined the epistatic effects between the lpr mutation and non-Fas susceptibility genes. QTLs are discussed in relation to previously described loci, with emphasis on their candidate genes.

Application of Candida solubilized cell wall beta-glucan in antitumor immunotherapy against P815 mastocytoma in mice.

This study was designed to evaluate the antitumor activity of CSBG, purified from the cell wall of Candida albicans IFO1385. First, as an effect of CSBG on P815 mastocytoma, significant prolonged survival and suppression of the tumor growth were observed. Second, the transfer of spleen cells from CSBG-sensitized BALB/c mice to CDF1 mice led to further suppression of tumor growth as well as P815-immunized spleen cells. Third, CSBG enhanced antitumor immunity in gene therapy using B7-1-transfected P815 cells. These results strongly suggest that CSBG enhances the host defense response to tumor due in part to an adjuvant effect.