Loss of H-2Kk gene product(s) from an AKR spontaneous leukaemia

Serological absorption analyses and immunochemical studies of the H-2 antigenic specificities on the AKR (H-2^k) spontaneous leukaemia K36 have been performed. The results confirm the absence of the H-2K^k gene product from the cell surface and from detergent solubilised tumour cells. A genetic mechanism is considered for this phenotypic alteration.     

Introduction of the H-2Dk gene into a class I-negative tumor cell line confers interferon-gamma inducibility upon the silent endogenous H-2Kk gene.

Kgv cells do not constitutively express class I mRNA or protein. Interferon (IFN)-gamma, but not IFN-alpha/beta, induces H-2Dk expression. IFN does not induce H-2Kk expression. We examined constitutive and IFN-inducible class I expression on Kgv cells stably transfected with genomic clones of H-2Kk or H-2Dk and on somatic cell hybrid lines constructed between Kgv cells and constitutively class I-positive cells of a distinguishable H-2 haplotype. Our results suggest that both the lack of constitutive class I expression and the inability of IFN-alpha/beta to induce class I expression on Kgv cells are primarily due to cis-regulatory mechanisms. However, stable introduction of the H-2Dk gene into Kgv cells conferred IFN-gamma inducibility upon the silent endogenous H-2Kk gene. Therefore, the failure of IFN-gamma to induce H-2Kk expression on Kgv cells is due, at least in part, to a trans-regulatory mechanism.     

Analysis of H-2-linked immune responses involved in resistance to AKR tumor growth

H-2-associated immune response gene(s) govern resistance to growth of a spontaneous AKR lymphoma, BW5147. The antigenic specificities recognized by the anti-BW5147 humoral response have been characterized and include: Thy-1, a T -cell differentiation antigen; gp70, a retroviral envelope protein; and several previously uncharacterized proteins, including a 78 000 molecular mass protein, p78, which is restricted to expression on BW5147 cells and five phosphoproteins with molecular masses of 33 000, 29 000, 23 000, 17 000, and 16 000. Only mice which are able to respond to Thy-1, p78, and the phosphoproteins can survive an inoculation of BW5147. Thus, resistance to BW5147 is complex and involves multiple antigens with possible roles in tumor rejection.Abbreviations used in this paper DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - Ir immune response - MuLV murine leukemia virus - NMS normal mouse serum - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Localization of H-2Kk in developing mouse teeth using monoclonal antibody

The in situ distribution of H-2 antigens during mouse tooth morphogenesis was investigated using monoclonal antibodies to H-2Kk and indirect immunofluorescent techniques. H-2 antigens were detected in the basement membrane region of fetal molars; they were absent from both the epithelial and dental mesenchyme. H-2 antigens were not found in newborn and 4-day-old mouse molars.     

DNA sequence analysis of the C3H H-2Kk and H-2Dk loci. Evolutionary relationships to H-2 genes from four other mouse strains

We generated nucleotide sequences for H-2Kk and H-2Dk from the C3H mouse, as well as for a genomic clone of H-2Db, in order to conduct an evolutionary analysis of the H-2 genes from three haplotypes, k, d, and b. H-2Kk from both the C3H and AKR strains, H-2Kd, H-2Kb, H-2Dk, H-2Ld, H-2Dd, H-2Db, and H-2Dp DNA sequences were aligned, and the alignments used to construct phylogenetic trees inferring the evolutionary relationships among the nine genes by two independent methods. Both approaches yielded trees with similar topologies. In addition, the sequence alignments revealed patterns of nucleotide substitutions which implicate both point mutation and recombination in the divergence of the H-2 genes. Future considerations for evolutionary analysis of class I genes are discussed.     

Cell surface expression of cytotoxic T lymphocyte-defined, AKR/Gross leukemia virus-associated tumor antigens by normal AKR.H-2b splenic B cells

We previously described a system in which H-2Kb-restricted C57BL/6 (B6) cytotoxic T lymphocytes (CTL) could be raised that were specific for tumors, such as the thymic lymphoma AKR.H-2b SL1, that were induced by endogenous AKR/Gross murine leukemia virus and that expressed the Gross cell surface antigen. In this study, certain normal lymphoid cells from AKR.H-2b mice were also found to express target antigens defined by such anti-AKR/Gross virus CTL. AKR.H-2b spleen, but surprisingly not thymus, cells stimulated the production of anti-AKR/Gross virus CTL when employed at either the in vivo priming phase or the in vitro restimulation phase of anti-viral CTL induction. This selective stimulation by spleen vs thymus cells was not dependent on the age of the mice over the range (3 to 28 wk) tested. Both AKR.H-2b spleen and thymus cells, however, were able to stimulate the generation of H-2-restricted B6 anti-AKR minor histocompatibility (H) antigen-specific CTL. Thus, AKR.H-2b spleen cells appeared to display the same sets (minor H and virus-associated) of cell surface antigens recognized by CTL as the AKR.H-2b SL1 tumor, whereas AKR.H-2b thymocytes were selectively missing the virus-associated target antigens, a situation analogous to that of cl. 18-5, a variant subclone of AKR.H-2b SL1 insusceptible to anti-AKR/Gross virus CTL. Like AKR.H-2b thymocytes, neither AKR spleen cells or thymocytes nor B6.GIX + thymocytes were able to stimulate the generation of anti-AKR/Gross virus CTL from primed B6 responder cell populations. In contrast, both T cell-enriched and B cell-enriched preparations derived from AKR.H-2b spleen cells were able to stimulate at the in vitro phase of induction, although B cell-enriched preparations were considerably more efficient. The discordant results obtained with AKR.H-2b spleen cells vs thymocytes were confirmed and extended in experiments in which these cells were employed as target cells to directly assess the cell surface expression of virus-associated, CTL-defined antigens. Thus, AKR.H-2b spleen cells, but not thymocytes, were recognized by anti-AKR/Gross virus CTL when fresh normal cells were tested as unlabeled competitive inhibitors, or when mitogen blasts were tested as labeled targets. Fresh or lipopolysaccharide-stimulated B cell-enriched spleen cells were as efficiently recognized as unseparated spleen cell preparations. Unexpectedly, fresh or Lens culinaris hemagglutinin-stimulated T cell-enriched spleen cell preparations, although susceptible to anti-minor H CTL, were almost as poor as targets for anti-viral CTL as were thymocytes. Together, these results demonstrate the H-2-restricted expression of CTL-defined, endogenous, AKR/Gross virus-associated target antigens by normal AKR.H-2b splenic B cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The H-2Kk MHC peptide-binding groove anchors the backbone of an octameric antigenic peptide in an unprecedented mode

A wealth of data has accumulated on the structure of mouse MHC class I (MHCI) molecules encoded by the H-2(b) and H-2(d) haplotypes. In contrast, there is a dearth of structural data regarding H-2(k)-encoded molecules. Therefore, the structures of H-2K(k) complexed to an octameric peptide from influenza A virus (HA(259-266)) and to a nonameric peptide from SV40 (SV40(560-568)) have been determined by x-ray crystallography at 2.5 and 3.0 A resolutions, respectively. The structure of the H-2K(k)-HA(259-266) complex reveals that residues located on the floor of the peptide-binding groove contact directly the backbone of the octameric peptide and force it to lie deep within the H-2K(k) groove. This unprecedented mode of peptide binding occurs despite the presence of bulky residues in the middle of the floor of the H-2K(k) peptide-binding groove. As a result, the Calpha atoms of peptide residues P5 and P6 are more buried than the corresponding residues of H-2K(b)-bound octapeptides, making them even less accessible to TCR contact. When bound to H-2K(k), the backbone of the SV40(560-568) nonapeptide bulges out of the peptide-binding groove and adopts a conformation reminiscent of that observed for peptides bound to H-2L(d). This structural convergence occurs despite the totally different architectures of the H-2L(d) and H-2K(k) peptide-binding grooves. Therefore, these two H-2K(k)-peptide complexes provide insights into the mechanisms through which MHC polymorphism outside primary peptide pockets influences the conformation of the bound peptides and have implications for TCR recognition and vaccine design.     

Mouse Hepa-1 tumor is rejected by H-2Db-restricted CTL despite decreased MHC class I antigen expression

It has recently been hypothesized that tumor cells with reduced levels of MHC class I antigens are more susceptible to NK-mediated lysis and are rejected by NK cells, whereas tumor cells with normal levels of class I are rejected by tumor-specific CTL. We have tested this hypothesis using a mouse hepatoma system. The Hepa-1 tumor is a spontaneous H-2Kb loss variant that arose from the BW7756 tumor, when BW7756 was adapted to growth in culture. Our studies have shown that despite the loss of H-2Kb antigen, Hepa-1 is not more susceptible to NK lysis than its H-2Kb-transfected variants. These studies also suggested that NK cells were not responsible for rejection of the Hepa-1 tumor. The Hepa-1 tumor, therefore, appears to contradict the hypothesized linkage of MHC levels and NK susceptibility. Because NK cells are not involved in immunity to this tumor, we have sought to identify the effector cell responsible for Hepa-1 rejection. Cytotoxic T lymphocyte assays demonstrate that in vitro, Hepa-1 cells are lysed by Hepa-1-specific H-2Db-restricted CD4-CD8+ T lymphocytes. Footpad assays demonstrate that in vivo, Hepa-1 rejection requires CD4+CD8- and CD4-CD8+ Hepa-1-primed splenocytes. These results indicate that immunity to Hepa-1 is T cell mediated. Hepa-1 is therefore an example of an unusual tumor in that down-regulation of MHC class I antigen expression is associated with increased CTL susceptibility.     

Resistance to cell-mediated cytotoxicity is correlated with reduction of H-2K gene products in AKR leukemia

AKR leukemia cell lines differing in the amount of H-2K and H-2D antigens expressed on the cell surface were used to assess cell-mediated immune responses in syngeneic mice against Gross/AKR murine leukemia virus (MuLV)-induced tumors. Leukemic cells with reduced expression of H-2K^k antigens were inactive as inducers of Gross-MuLV/H-2^k-specific cytotoxic T lymphocytes (CTL) and resistant to lysis by CTL raised against H-2K^k positive AKR leukemia cells. H-2K^k positive leukemias induced cytotoxic effectors, which upon restimulation in vitro, lysed the stimulating and other H-2K^k positive leukemia cells. In antibody inhibition experiments, T-cell-mediated cytotoxicity to these leukemias could only be inhibited by antisera and monoclonal antibodies specific for the H-2K^k antigens. Due to this specific role of H-2K^k antigens in T-cell cytotoxicity to Gross/AKR MuLV-induced tumors, reduced expression of H-2K^k antigens on spontaneous AKR leukemic cells could have important implications for surveillance of these neoplastic cells.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MuLV murine leukemia virus     

Induction of endogenous avian tumor virus gene expression by pyrrolizidine alkaloids

The pyrrolizidine alkaloids (PA) are toxic compounds which occur naturally in plant species throughout the world. They have been implicated as both carcinogenic and mutagenic agents. An active metabolite of the alkaloids, the pyrrole, which is a strong alkylating agent, is thought to be the toxicant. The naturally occurring alkaloid, jacobine , is able to induce the production of endogenous avian RNA tumor virus particles in cultured chick embryo fibroblasts (CEF). When jacobine was modified to form retronecine it no longer induced virus particles. Conversion of retronecine to its pyrrole resulted in a compound capable of inducing virus particle production. The isobutyryl monoester of retronecine was also able to induce virus particle production, but the isobutyryl monoester pyrrole was unexpectedly inactive as an inducer. This type of viral induction system is useful for studying the effect of modification of the inducer on its biological activity.     

DAC can restore expression of NALP1 to suppress tumor growth in colon cancer

Despite recent progress in the identification of genetic and molecular alternations in colorectal carcinoma, the precise molecular pathogenesis remains unclear. NALP1 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 1) is a member of the nucleotide-binding oligomerization domain-like receptor family of proteins that are key organization proteins in the inflammasome. It is reported that NALP1 plays a central role in cell apoptosis, pyroptosis, inflammatory reactions and autoimmune diseases. DAC (5-aza-2-deoxycytidine) is an antitumor drug useful to lung cancer, myelodysplastic disorders, myelodysplasia and acute myeloid leukemia. In this study, we examined the expression of NALP1 in human normal and cancerous colon tissues using tissue microarray, western blot and quantitative real-time PCR and we measured the expression of NALP1 in three kinds of colon cancer cell lines and animal models before and after treatment with DAC. Furthermore, we examined the treatment effects of DAC on colon cancer in our animal model. Our data indicate that NALP1 is expressed low in human colorectal tumoral tissues relative to paratumoral tissues and was associated with the survival and tumor metastasis of patients. The expression of NALP1 increased after treatment with DAC both in vitro and in vivo. Furthermore, DAC suppressed the growth of colon cancer and increased lifespan in mouse model. Therefore, we conclude that NALP1 is expressed low in colon cancer and associated with the survival and tumor metastasis of patients, and treatment with DAC can restore NALP1 levels to suppress the growth of colon cancer.Colorectal cancer is one of the most common forms of fatal cancer in the world,¹ yet the molecular mechanisms underlying its growth are poorly understood.² Intestinal epithelial cells play an important role in the innate defense of the intestine, and impairment of epithelial functions can result in inflammatory bowel diseases (IBD)^{3, 4, 5, 6} such as ulcerative colitis. Epidemiological studies in patients with IBD have clearly identified chronic active inflammation as a major risk factor for colon adenocarcinoma.^{7, 8, 9} Although colitis-associated colorectal cancers (CACs) comprise ^<5% of all colorectal cancers,¹⁰ the cumulative incidence of CAC in patients with ulcerative colitis 25–30 years after diagnosis ranges from 8 to 43%, accounting for one-sixth of all deaths in this group.¹¹ Treatment with nonsteroidal anti-inflammatory therapy reduces the risk of cancer in ulcerative colitis patients by 40–50% and reduces the risk of developing polyps in patients with familial adenomatous.^{12, 13} The data suggest that anti-inflammatory therapy with nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of CAC.¹⁴The NALP1 (nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 1) protein, which contains a pyrin domain at its NH3 terminus and a CARD (caspase recruitment domain-containing protein) domain at its COOH terminus, has attracted recent interest in the field of CAC. NALP1 (also called CARD7 or NAC) is the first NALP-family protein to be discovered by its sequence homology to APAF-1 (apoptotic protease-activating factor-1) and has been implicated in cell responses to apoptotic and inflammatory stimuli. NALP1 is a multidomain scaffold protein that contains an N-terminal pyrin domain (PYD) followed by a centrally located NACHT domain, five tandem LRR domains, a FIIND (domain with function to find) domain and a C-terminal CARD domain.¹⁵ NALP1 protein is believed to interact with APAF-1 to subsequently enhance apoptosis and/or activate the proinflammatory caspases in conjunction with ASC (apoptosis-associated speck-like protein containing a CARD). ASC is an essential component of the inflammasome and connects NALP1 to caspase-1. The PYD of ASC interacts with the PYD of NALP1, and the CARD of ASC recruits the CARD of procaspase-1. Furthermore, NALP1 has been implicated in cell pyroptosis, a newly discovered form of programmed cell death.¹⁶The 5-aza-2-deoxycytidine (DAC) is a DNA methylation inhibitor that has found use as an antitumor drug in mammary cancer,¹⁷ non-small-cell lung cancer,¹⁸ rhabdomyosarcoma and medulloblastoma.¹⁹ At low doses, DAC can exert durable antitumor effects without cytotoxicity both in vitro and in vivo.^{18, 20}Recent articles have focused on the role of NALP1 in pyroptosis and inflammation, but the expression of NALP1 in colon cancer remains unknown. Furthermore, studies examining the effects of DAC on colon cancer are rare. Considering the specific multidomain architecture of NALP1, the abnormal expression of NALP1 in some diseases and its signal-mediated role in apoptosis, we choose human colorectal carcinoma tissue as a representative example of clinical solid tumors and examined the expression of NALP1. We designed our experiment to explore the correlation between NALP1 and human colorectal carcinoma. We studied the expression of NALP1 in human normal colon tissues and colon cancer tissues and measured the expression level of NALP1 in three kinds of colon cancer cell lines in the presence or absence of DAC treatment to investigate the mechanisms underlying reduced expression of NALP1 in colon cancer and the effects of DAC treatment.     

Tissue-specific expression of the mouse Q10 H-2 class-I gene during embryogenesis

We have studied the pattern of expression of the Q10 gene, a H-2 class-I gene located in the major histocompatibility complex which encodes a soluble class-I molecule, in the mid-gestation mouse embryo, and compared it to those of two other class-I genes, namely Kd and 37, the latter gene located in the thymus leukemia region. We found that the steady-state amount of these different mRNAs gradually increased from day 13 to day 18. By comparison with the level of expression of these genes in adult liver, the increase during gestation was fairly more marked for Q10 mRNA than for the others. Furthermore, we found that the Q10 gene is transiently expressed in the endoderm layer of the visceral yolk sac and in the fetal heart. Expression in the latter tissue decreases abruptly while increasing in the liver. It has been proposed that the Q10 protein is involved in immune tolerance. However, the time course of expression of Q10 mRNA and its tissue distribution during embryogenesis suggest that the Q10 protein could play a role in the differentiation of hematopoietic stem cells.     

Interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. II. Levels of expression of H-2K, H-2D, and H-2L in different mouse strains

The numbers of MHC class I molecules expressed by spleen cells from various mouse strains were determined by using MHC-specific monoclonal antibodies and a radioactive binding assay. Although small differences were found to exist in some cases, our general conclusion is that different mice of the same strain, congenic mice of different haplotypes, and syngeneic mice of varying background all express similar numbers of class I antigens. B10.A mice (8 to 10 wk old), for example, express 5.3 X 10(4) Kk molecules/cell, 5.4 X 10(4) Dd molecules/cell, and 2.2 X 10(4) Ld molecules/cell. Some of the differences observed in class I antigen expression included: 1) the level of Kk expression increased to a small but significant extent with age in B10.A mice; 2) female B10.A mice expressed slightly higher amounts of Kk than male mice; and 3) B10.A(2R) and B10.A(4R) recombinant strains expressed elevated levels of K-end antigens and slightly decreased levels of D-end antigens when compared with the unrecombinant B10.A strain. In several strains, F1 mice express approximately 50% as many copies of each parental antigen as do the homozygous parents. B10 mice, which are negative for the L antigen, nevertheless express the same total number of D-end molecules as do B10.A mice. The data suggest that the levels of expression of MHC class I molecules are controlled by at least two factors: gene dosage and another factor(s) that gives rise to the small variations in class I antigen expression seen with age, sex, and strain, and to the low expression of Ld relative to Dd and Kk.     

The regulation of expression of mouse mammary tumor virus DNA by steroid hormones and growth factors

Mouse mammary tumor virus (MMTV) expression is associated with hyperplastic alveolar growth and subsequent development of mammary cancers in the mouse. The expression of this virus is also controlled by factors involved in the normal proliferation and differentiation of the mammary epithelium. During pregnancy when the mammary gland undergoes massive proliferation, MMTV expression is increased. Steroid hormones and growth factors that play an important role in the proliferation of mammary gland cells are responsible for the increased MMTV expression. In sarcomatous transformation of mouse mammary epithelial cells, MMTV expression is repressed. This repression is due to negative control of MMTV expression by transforming growth factor-beta (TGF beta). This growth factor is produced in high amounts when mammary epithelial cells progress into the transformed state. The expression of MMTV is therefore under multiple control by steroid hormones and growth factors.     

Identification of mouse serum miRNA endogenous references by global gene expression profiles

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments.