Beta 2m mRNA Research Articles

Presence of minute cancer cell dissemination in peritoneal lavage fluid detected by reverse transcription PCR is an independent prognostic factor in patients with resectable pancreatic cancer

Presence of minute cancer cell dissemination in peritoneal lavage fluid detected by reverse transcription polymerase chain reaction (RT-PCR) has been reported to be a reliable predictor of the prognosis in several kinds of cancers, but has not been determined in pancreatic cancer. Peritoneal lavage fluid was harvested just after a laparotomy in 83 patients with adenocarcinoma of the pancreas. Half of the fluid was examined by cytology and the remaining half was used to measure carcinoembryonic antigen/beta-2-microglobulin (beta2M) mRNA expression. Patients were followed after surgery to evaluate its clinical significance. Among 83 patients, 3 were cytologically positive (CY+), while 23 were positive by RT-PCR (PCR+). Seventy-one patients underwent a surgical resection whereas 12 were unresectable. Because 2 were CY+ among the 71 operated patients, the remaining 69 CY- patients were further investigated. Among those 69 patients, PCR+ was observed in 15 patients, whose incidence of postoperative peritoneal recurrence was significantly higher than that in PCR- patients (21% vs 4% at 3 years; P = .039). Moreover, both the recurrence-free rate in the abdominal cavity (peritoneal or local recurrence, excluding liver metastases) and the overall survival rate were better in PCR- patients than PCR+ patients (78% vs 33%, P = .0045 and 67% vs 46%, P = .0151). A multivariate analysis revealed positive lymph node metastases (hazard ratio; 5.18) and positive RT-PCR (hazard ratio; 3.65) were independent prognostic factors. The RT-PCR-based cancer cell detection was an independent prognostic factor in patients with resectable adenocarcinoma of the pancreas and had close association with local or peritoneal recurrence.

Synthesis of beta(2)-microglobulin-free, disulphide-linked HLA-G5 homodimers in human placental villous cytotrophoblast cells.

Human leucocyte antigen-G (HLA-G) is a natural immunosuppressant produced in human placentas that binds differently to the inhibitory leucocyte immunoglobulin-like receptors LILRB1 (ILT2) and LILRB2 (ILT4) according to its biochemical structure. To predict the binding functions of the HLA-G5 soluble isoform synthesized in placental villous cytotrophoblast (vCTB) cells, we investigated structural features of this protein. Biochemical and immunological studies showed that vCTB cell HLA-G5 heavy (H)-chain proteins are disulphide-bonded homodimers unassociated with beta(2)-microglobulin (beta2m) light-chain proteins. Although comparatively low levels of beta2m messenger RNA (mRNA) were identified by real-time reverse transcription-polymerase chain reaction, immunoprecipitation studies failed to detect beta2m protein even when specific mRNA was doubled by transduction of a lentivirus-beta2m complementary DNA into vCTB cells. No abnormalities were identified in the translational start codon of vCTB cell beta2m mRNA and differentiation into syncytium did not promote beta2m synthesis. The failure of vCTB cells to exhibit beta2m in vitro was paralleled by a lack of detectable beta2m in vCTB cells in vivo. Lack of the beta2m protein could be the result of low levels of beta2m transcripts or of as yet unidentified translational defects. Experiments with recombinant ectodomains of LILRB indicate that beta2m-free HLA-G binds strongly to LILRB2, a receptor that is expressed by macrophages. This potentially immunosuppressive cell type is abundant in the pregnant uterus. Thus, our findings are consistent with the postulate that the natural beta2m-free homodimeric form of HLA-G5 synthesized in primary vCTB cells could comprise a particularly effective tolerogenic molecule at the maternal-fetal interface.

Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers

To establish a novel molecular diagnostic method of detecting circulating tumor cells (CTCs) LS174T colon cancer cells were serially diluted with normal blood. Additional peripheral blood samples were collected from 25 patients with colorectal carcinoma. Mononuclear cells (MNCs) were collected, equally divided into four parts, and then cancer cells were enriched by four methods: method A, nonimmunobead method; method B, negative immunobead method: CD45 immunomagnetic beads were used to deplete the leukocytes; method C, positive immunobead method: Ber-EP4 immunomagnetic beads were used to enrich cancer cells; method D, negative-and-positive immunobead method: CD45 immunomagnetic beads were first used to deplete the leukocytes from MNC and then Ber-EP4 immunomagnetic beads were used to enrich cancer cells. Finally, real-time quantitative RT-PCR was used to monitor mRNA expression of beta2-mircoglobulin (beta2M) and carcinoembryonic antigen (CEA). The relative CEA mRNA values were corrected with reference to beta2M mRNA, to CEA mRNA/beta2M mRNA ratios according to a CEA mRNA external standards prepared with tenfold serial dilutions (1-10(4) IS174T cells) of cDNA and beta2M mRNA external standards prepared with tenfold serial dilutions (10(2)-10(7) leukocytes) of cDNA. In recovery experiments a significant correlation between the number of cancer cells and CEA mRNA expression was found when CD45 or Ber-EP4 immunomagnetic beads were used alone. A highly significant correlation was found when CD45 and Ber-EP4 immunomagnetic beads were used successively. The sensitivity of method D was one cancer cell per milliliter of blood. Circulating cancer cells were detected in 19 of 25 patients with colorectal cancers. The relative CEA mRNA value obtained by method D was the smallest. The positive detection rate of circulating cancer cells in patients at Dukes' B, C, and D stages were 25.0% (1/4), 83.3% (10/12), and 88.9% (8/9). Combinative use of immunomagnetic isolation followed by real-time RT-PCR is a useful technique to detect circulating tumor cells in patients with colorectal carcinomas. Applying negative and positive immunomagnetic beads successively yields the highest correlation with amount of tumor cells.

Total loss of MHC class I in colorectal tumors can be explained by two molecular pathways: β2‐microglobulin inactivation in MSI‐positive tumors and LMP7/TAP2 downregulation in MSI‐negative tumors

The mechanisms that lead to loss of MHC class I expression in different types of tumors are not yet fully known. Accordingly, we studied colorectal carcinomas to elucidate the specific mechanisms of evasion of the T-cell immune response. We selected tumors with total loss of MHC class I expression and studied 124 colorectal carcinomas with immunohistochemical staining and anti-HLA monoclonal antibodies (mAb). Fourteen of 124 (11%) tumors exhibited a phenotype with HLA class I total loss. Microsatellite instability (MSI) analysis was also carried out in the same tumor samples. The expression of beta2-microglobulin (beta2m), HLA-A, B, and C antigens, transporter associated with antigen processing 1 (TAP1), TAP2, low-molecular-weight protein 2 (LMP2), and LMP7 were analyzed using reverse-transcription polymerase chain reaction (RT-PCR) in microdissected tumor samples. Four of 14 microsatellite instability-positive (MSI+) and W6/32 mAb-negative tumors showed biallelic inactivation of beta2m and accumulation of HLA class I heavy chain in the cytoplasm. MSI-negative (MSI-)/W6/32 mAb-negative tumors presented alterations in the expression of components of the antigen processing machinery (APM). Nine of 10 tumor samples showed LMP7 gene downregulation, and four of 10 presented TAP2 dysregulation. This group apparently expressed normal levels of heavy chain and beta2m mRNA. Two major mechanisms in colorectal cancer appear to be responsible for the total loss of MHC surface expression (beta2m mutations and LMP7/TAP2 downregulation) that may contribute to the failure of T lymphocyte recognition during an immune response. The precise identification of the molecular defects that underlie HLA class I abnormalities will have important implications for patients receiving T-cell-based specific immunotherapy.

Expression of β2-microglobulin and c-fos mRNA: Is there an influence of high- or low-flux dialyzer membranes?

Dialysis-related amyloidosis is an important complication of long-term hemodialysis (HD) therapy with several pathogenetic factors. One of them is the influence of the dialyzer membrane type on the synthesis of beta2-microglobulin (beta2m). In vitro results are controversial. Thus, the hypothesis of whether in vivo beta2m generation is induced by the HD procedure and whether this induction depends on the type of the used dialyzer membrane should be tested. The aim of the present study was to investigate the influence of "biocompatible" high-flux versus "bioincompatible" low-flux HD on in vivo beta2m generation as well as the induction of the early activation gene c-fos in peripheral blood cells. Six nondiabetic HD patients [mean age 46 (21 to 69) years; Kt/V> 1.2] were included in a randomized crossover study using either a low-flux (cellulosic/cuprophan) or a high-flux (polyamide) dialyzer membrane. At the end of a four-week run-in period for each membrane, whole blood samples were taken before, immediately at, and four hours after the end of the dialysis session. MRNA was extracted, and after transcription to cDNA, quantitative polymerase chain reaction was performed for the beta2m gene, the early response gene c-fos, and the GAP-DH housekeeping gene. Based on the applied method for detection of specific mRNA, the results were given as ratio of beta2m or c-fos cDNA per GAP-DH cDNA. General cell activation during HD was indicated by increasing mRNA expression of c-fos related to the time course of the dialysis session, whereas beta2m did not change significantly. However, no difference was found when comparing the low-flux and the high-flux dialyzer membranes. Despite the evidence for activation of peripheral blood cells, as indicated by increasing c-fos message, no sign of beta2m mRNA induction during HD procedure with different dialyzer membranes was seen. Our results suggest that there is post-transcriptional regulation of beta2m generation and/or release as well as the influence of the dialyzer membrane type on post-translational processes, that is, advance glycation end products (AGE) or conformational modification of the beta2m protein. Furthermore, our data demonstrate that gene expression patterns during dialysis and/or uremia are not homogenous and need to be investigated further, especially with respect to the proinflammatory role of early leukocyte activation signals.

Regulation of β2-microglobulin expression in different human cell lines by proinflammatory cytokines

Proinflammatory monocytic cytokines such as interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) and IL-6 have been incriminated in the pathogenesis of elevated beta2-microglobulin (beta2M) serum concentrations in patients undergoing haemodialysis with so-called bioincompatible dialyser membranes. However, neither the source of the elevated serum beta2M nor the precise role of monocytic cytokines in the expression of the beta2M gene have been elucidated conclusively. The aim of the current study was to evaluate whether monocytic cytokines, and in particular IL-6, are regulators of beta2M gene expression in human hepatoma cells, T-lymphocytes and monocytes. HepG2 and HuH7 human hepatoma cells, Jurkat T-cells, monocytic MonoMac6 cells, primary human monocytes and synoviocytes were stimulated with IL-1beta, IL-6, interferon-alpha (IFN-alpha), IFN-gamma or conditioned media from lipopolysaccharide (LPS)-treated monocytes. Expression of beta2M mRNA was analysed by Northern blotting, beta2M protein synthesis was determined by metabolic labelling and immunoprecipitation, and beta2M secretion was measured by an enzyme-linked immunosorbent assay (ELISA). In all cell types tested, IFN-gamma and, to a lesser extent, IFN-alpha stimulated gene expression of beta2M resulting in an increased synthesis and secretion of beta2M protein. Neither IL-1beta and IL-6 nor supernatants from LPS-treated monocytes were capable of inducing beta2M gene expression, with the exception of a small increase in HuH7 hepatoma cells upon IL-1beta treatment. The present study provides evidence that interferons are important regulators of beta2M expression. It also shows that proinflammatory monocytic cytokines do not modulate directly the expression of beta2M in cells of hepatic, monocytic and T-lymphocytic origin. Whether they influence beta2M synthesis and secretion indirectly by modulating interferon synthesis needs to be elucidated.

MHC class I genes of the channel catfish: sequence analysis and expression.

Four cDNAs encoding the major histocompatibility complex (MHC) class I alpha chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I alpha chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I alpha and beta2-microglobulin (beta2m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 Mr glycoprotein (presumably class I alpha) was associated with beta2m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of beta2m with the MHC class I alpha chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts.

Molecular mechanisms of loss of beta 2-microglobulin expression in drug-resistant breast cancer sublines and its involvement in drug resistance.

In this study, we investigated the mechanism of the loss or decreased expression of beta 2-microglobulin (beta 2m) in several drug-resistant sublines of MCF-7 and in a doxorubicin (DOX)-resistant variant of the T-47D breast cancer cell line. beta 2m protein and RNA are not expressed in highly metastatic, multidrug-resistant MCF-7/Adr cells with high resistance to DOX. Nuclear run-on transcription and RNA stability assays demonstrate that while beta 2m in MCF-7/Adr cells is transcribed, its mRNA is rapidly degraded after synthesis in these cells, indicating that it is controlled by post-transcriptional mechanisms. We also show that an MCF-7 subline (MCF-7/Adr-5) expressing a very low level of resistance to DOX has a decreased level of beta 2m expression. Treatment with actinomycin D revealed that the half-life of beta 2m mRNA in MCF-7 and MCF-7/Adr-5 cell lines was comparable. Nuclear run-on transcription analysis revealed a decreased rate of beta2m transcription in MCF-7/Adr-5 cells compared to that in MCF-7 cells. Moreover, beta 2m mRNA remained undetectable in MCF-7/Adr cells following cycloheximide treatment. However, in MCF-7 cells, increased beta 2m mRNA was observed after 12 h, and a similar level of increased mRNA expression was observed after 36 h of cycloheximide treatment in MCF-7/Adr-5 cells; these results suggest that one of the mechanisms controlling beta 2m mRNA expression might be a negative regulatory protein in MCF-7/Adr-5 cells. Analysis of the beta 2m status of other drug-resistant MCF-7 sublines revealed that deregulation of beta 2m is not limited to DOX resistance, but can also be detected in cells selected for resistance to mAMSA and DOX-verapamil. In addition, our data show that reduced beta 2m expression correlates with the decreased levels of estrogen receptor (ER) expression in the DOX-resistant MCF-7/Adr and T-47D/Adr-4 human breast cell lines. Furthermore, we provide evidence that the partial inhibition of beta 2m by antisense RNA results in 2-3-fold decreased sensitivity of MCF-7 cells to DOX and mAMSA. Moreover, the addition of exogenous beta 2m protein near its physiological human serum concentration can modulate the DOX sensitivity of the MCF-7 antisense beta 2m and control transfectants. Therefore, these results indicate that lost or decreased beta 2m expression is involved in the development of the drug-resistant phenotype and correlates with the loss of ER in human breast cancer cell lines.

Beta2-Microglobulin mutations, HLA class I antigen loss, and tumor progression in melanoma.

The potential negative impact of HLA class I antigen abnormalities on the outcome of T cell-based immunotherapy of melanoma has prompted us to investigate the mechanisms underlying lack of HLA class I antigen expression by melanoma cell lines Me18105, Me9923, and Me1386. Distinct mutations in the beta2-microglobulin (beta2m) gene were identified in each cell line which result in loss of functional beta2m. In Me18105 cells, an aberrant splicing mechanism caused by an A--> G point mutation in the splice acceptor site of intron 1 of the beta2m gene, deletes 11 bp from the beta2m mRNA creating a shift in the reading frame. In Me9923 cells a 14-bp deletion in exon 2 and in Me1386 cells a CT deletion in exon 1 of the beta2m gene produce a frameshift mutation. The beta2m gene mutations identified in Me18105, Me9923, and Me1386 cells were also detected in the surgically removed melanoma lesions from which the cell lines originated. Transfection of each melanoma cell line with a wild-type beta2m gene restored HLA class I antigen expression and, in Me18105 cells, recognition by Melan-A/MART-1-specific, HLA-A2-restricted cytotoxic T lymphocytes. Interestingly, the beta2m mutation present in Me9923 cells that were derived from a metastatic lesion was also found in the Me9923P cell line that originated from the autologous primary lesion. These data suggest that beta2m mutations in melanoma cells may be an early event in progression to the malignant phenotype.

The tetraspanin protein CD82 associates with both free HLA class I heavy chain and heterodimeric beta 2-microglobulin complexes.

CD82 is a tetraspan transmembrane protein on NK/LAK-susceptible targets. A single highly glycosylated protein of heterogeneous molecular mass (50-90 kDa) was immunoprecipitated by anti-CD82 from Nonidet P-40 lysates of various B cell lines, Raji, Daudi, 721, and 721.134. Using the milder detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), additional proteins were coprecipitated with CD82 from surface iodinated B cell lines, including a major band at 45 kDa, identified as the HLA class I heavy chain by sequential immunoprecipitations and sequential immunoprecipitation-Western blot analysis. Cocapping experiments confirmed the molecular association of CD82 and HLA class I at the cell surface of these B cell lines. CD82 could be coprecipitated with both mature and beta 2-microglobulin (beta 2m)-free heavy chains of MHC-I from CHAPS extracts. No association between MHC-I and CD82 was found in the beta 2m-deficient Daudi cell line or after co-in vitro translation of CD82, MHC heavy chain, and beta 2m mRNA. The most likely source of free class I heavy chains at the cell surface is by dissociation of beta 2m-associated class I molecules. These results suggest that association of CD82-MHC-I takes place at the cell surface and could interfere with the capacity of the MHC-I complex to protect targets from NK-mediated cytotoxicity.

Expression of distinct conformations of free HLA-Cw4 heavy chains in transfected neuroblastoma cells.

Epitope mapping of HLA-Cw4 indicates that the two monoclonal antibodies (mAbs) L31 and M38, specific for beta 2-microglobulin (beta2m)-free HLA-C heavy chains, react preferentially with the KYK motif, located in the binding groove (alpha1 domain). Transfection of HLA-Cw4 cDNA into a neuroblastoma cell line, which normally expresses negligible HLA class I, resulted in the constitutive surface expression of molecules displaying different reactivities with the two mAbs. This cellular system was used to determine whether L31 and M38 recognize distinct conformations of beta2m-free HLA-C proteins, and to investigate their mechanism of expression. Interferon-gamma greatly enhanced the expression of L31-reactive free chains, while abolishing that of M38-reactive molecules. The cytokine-induced expression of L31-reactive molecules was inhibited by anti-sense oligonucleotides specific for beta2m mRNA, while constitutive expression of L31-reactive molecules was only partially affected. Exogenous beta2m resulted in a reduction of constitutive L31 reactivity, and in a concomitant increase of M38 reactivity. These results indicate that: 1) at the cell surface, L31 and M38 react with two distinct conformations of HLA-Cw4 beta2m-free heavy chains, of which the L31-reactive conformation is the least folded; 2) the expression of both conformers can be modulated by endogenous or exogenous beta2m; and (3) L31-reactive molecules exposed at the cell surface are likely to derive from the dissociation of empty HLA-Cw4/beta2m complexes.

Potential effect of metabolic acidosis on beta 2-microglobulin generation

Beta 2-microglobulin (beta 2M) is responsible for dialysis-associated amyloidosis. Level of beta 2M in plasma increase during chronic renal failure; however, retention does not appear to be the sole mechanism responsible. The effect of metabolic acidosis on beta 2M production was examined. Thirty-six patients with stable chronic renal insufficiency, 12 uremic patients before their first dialysis, 8 hemodialysis patients who were assigned to acetate or bicarbonate dialysate and then crossed over to the alternative regimen, and 6 normal subjects given NH4Cl to initiate metabolic acidosis were studied. In vitro studies in the human myeloid cell line U 937 were also performed. beta 2M protein was measured with ELISA, beta 2M mRNA was measured with reverse transcription polymerase chain reaction, and the U 937 cells were studied at two pH levels with FACScan flow cytometry. The cells were exposed in vitro up to 60 min in a buffered incubation medium to either pH 5.10 or pH 7.34. An inverse correlation was found between beta 2M and bicarbonate concentrations in plasma in the stable chronic renal failure patients (r = -0.54; P < 0.05) and in the uremic patients before their first dialysis (r = -0.72; P < 0.05). In hemodialysis patients, blood pH and plasma bicarbonate values were lower (P < 0.05) and beta 2M concentrations in plasma were higher (P < 0.05) with acetate than with bicarbonate dialysate. In normal men, NH4Cl resulted in an increase (P < 0.05) in beta 2M mRNA expression in lymphocytes by an average factor of 1.5 (range, 1.1 to 1.8). In U 937 cells, the cell surface expression of beta 2M and HLA Class I heavy chain assembled with beta 2M decreased at low pH compared with normal pH. Concomitantly, an increase in beta 2M release into the supernatant was observed, possibly as the result of beta 2M dissociation from cell surface HLA Class I complex. The results suggest that metabolic acidosis may enhance cellular beta 2M generation and release.

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels.

Distinct patterns of expression of MHC class I and beta 2-microglobulin transcripts at early stages of mouse development.

The class I surface Ag recognized during tissue graft rejection are composed of a 40- to 45-kDa H chain and a 12-kDa L chain, beta 2-microglobulin (beta 2m). Regulation of MHC gene expression during early development is thought to play an essential role in maternal tolerance of the fetal allograft. Here we used in situ hybridization techniques to characterize the temporal and spatial pattern of expression of MHC class I mRNA in the developing mouse embryo. MHC class I transcripts were initially detected at day 9.5 postcoitus in the primary and secondary trophoblast giant cell populations. At this stage, none of the remaining fetal components of the developing placenta expresses MHC class I mRNA. By contrast, the outer zone of trophoblast giant cells is the only trophectoderm-derived tissue in the developing placenta that does not express detectable levels of beta 2m mRNA. These findings indicate that the onset of MHC class I and beta 2m gene expression in early postimplantation stage embryos is not coordinately regulated. In addition, we made use of pluripotent embryonic stem (ES) cells to study developmental regulation of MHC class I and beta 2m gene expression in vitro. ES cells can be induced to form endoderm-like cells by the technique of suspension culture. In contrast to retinoic acid-treated F9 cells, the endoderm derived from ES cells expressed high levels of beta 2m mRNA, but no detectable MHC class I transcripts. These findings are consistent with results of our in situ hybridization experiments showing high levels of expression of beta 2m mRNA, and extremely weak expression of MHC class I transcripts in derivatives of the visceral endoderm in vivo.

Stimulation of beta2-microglobulin synthesis in lymphocytes after exposure to Cuprophan dialyzer membranes

We tested beta 2-microglobulin (beta 2m) synthesis by peripheral blood lymphocytes of non-uremic donors after contact with dialyzer membranes. Under serum-free conditions, lymphocytes were incubated with different dialyzer membranes for four hours. After subsequent culture in the absence of membranes to yield an overall culture time of 24 hours, RNA was extracted. Messenger RNA for beta 2m was quantitated by Northern blotting with specific probes. Increased beta 2m mRNA was seen after a minimum of one hour contact time with Cuprophan both in the presence and absence of cycloheximide. While no consistent stimulation was seen with polyacrylonitrile or polycarbonate-polyether membranes, respectively, the stimulation of beta 2m mRNA with Cuprophan was of the same order of magnitude as that obtained with interferon gamma. Stimulation of beta 2m mRNA by Cuprophan was stereospecifically diminished by 5 mM L-fucose. Apart from stimulated transcription of beta 2m we could also show increased surface expression of MHC class I molecules, using FACS technique. The results further prove complement-independent blood cell activation by Cuprophan membranes. It is uncertain whether the results are relevant for beta 2m amyloidosis.

Selective loss of beta 2-microglobulin mRNA in human colon carcinoma.

Expression of MHC class I antigens requires the intracellular assembly of HLA-A,B,C H chains and beta 2-microglobulin (beta 2m). We have investigated the expression of free H chain, beta 2m, and their mRNAs in tissue sections of colon carcinomas that were defective for the native two-chain molecule. In all of these tumors, beta 2m protein and mRNA were found to be completely absent from the neoplastic cells, whereas free H chains and their respective mRNAs were present in abundance in the tumor cell cytoplasm. The selective abrogation of beta 2m expression represents a unique mechanism leading to a complete loss of class I antigen surface expression in vivo.

Crosses of two independently derived transgenic mice demonstrate functional complementation of the genes encoding heavy (HLA-B27) and light (beta 2-microglobulin) chains of HLA class I antigens.

In man a number of diseases are associated with certain alleles of MHC antigens. The most pronounced example is ankylosing spondylitis, which is strongly associated with HLA-B27. As a first step towards a model system to study the basis of this association, transgenic mice were generated that showed cell surface expression of the HLA-B27 antigen biochemically indistinguishable from HLA-B27 antigen expressed on human cells. This result was obtained by crossing two independently derived strains of mice, one of which is transgenic for the HLA-B27 heavy chain gene, and the other carrying and expressing the human beta 2m gene. Examination of HLA-B27 and human beta 2m mRNA in various tissues shows the two genes to be expressed in a coordinate fashion. The mRNA levels follow those of endogenous H-2 Class I genes.

Control of synthesis and expression of H-2 heavy chain and beta-2 microglobulin in AKR leukemias

In a series of newly isolated AKR leukemias, some tumors expressed large amounts of both H-2K and H-2D molecules, while others had reduced levels of both antigens. The number of H-2 antigens expressed in steady state showed a consistent correlation with the rate of synthesis of the H-2 class I heavy chain and the beta-2 microglobulin (beta 2m) light chain, and with the amount of H-2- and beta 2m-specific mRNA present in the tumors. Stimulation of leukemic cells with interferon induced an increased transcription of both H-2 and beta 2m mRNA. These results suggest that there is a mechanism that regulates, at the transcriptional level, the coordinate expression of H-2K and H-2D heavy chains, and the beta 2m light chains encoded by genes on chromosomes 17 and 2, respectively.

Asynchronous regulation of mouse H-2D and beta-2 microglobulin RNA transcripts.

The major transplantation (or H-2) antigens in the mouse are cell-surface glycoproteins composed of a heavy chain and a light chain, the beta-2 microglobulin (beta 2m). The expression of these proteins is regulated during development. Embryonic cells at early stages of development do not express these proteins. On the other hand, these molecules are present on the surface of all adult somatic cells. We investigated whether the expression of both chains was coordinately regulated. Using specific single-stranded DNA probes in an S1 nuclease analysis, we compared the relative amounts of H-2D and beta 2m transcripts in normal tissues, in transformed cells, and during embryonic development. Our results show that (1) the steady state level of beta 2m transcripts varies from one adult organ to another, while that of H-2D transcripts stays approximately the same; (2) upon transformation, the amount of H-2D-specific mRNA increases drastically, while the beta 2m mRNA level remains constant; (3) whereas the quantity of beta 2m mRNA increases during early development, the amount of H-2D mRNA remains at a very low level. These data suggest that the regulation of H-2D and beta 2m genes are not identical and that their activation during development is not synchronous.

Molecular analysis of an EL4 cell line that expresses H-2Db but not H-2Kb or beta 2-microglobulin.

EL4/Mar is a variant cell line that expresses H-2Db but neither H-2Kb nor beta 2-microglobulin (beta 2m). Southern and RNA blot analysis and immunoprecipitation of metabolically labeled proteins established that the B2m gene(s), beta 2m mRNA, and beta 2m protein are normal in this cell line. Somatic cell hybridization showed that the defect in this cell line was in the synthesis of H-2Kb, and RNA blot analysis with an H-2Kb specific oligonucleotide established that the H-2Kb gene(s) in this cell line was not transcribed into a stable mRNA species. The apparent absence of beta 2m on the surface of this cell line suggests that there may be some feature of the H-2Db molecule that allows it to be expressed in the absence of detectable beta 2m.