Comparison of HLA-G and MMP transcription in human tumor cell lines.

HLA-G antigens and matrix metalloproteinases (MMPs) expressed in various tumors are involved in tumor growth and metastasis. In this study, we investigated if correlation between HLA-G and MMP expression exists in different cell lines. We examined MMP transcription in two choriocarcinoma cell lines: JEG-3 (HLA-G positive) and JAR (HLA-G negative). We discovered that both cell lines express a similar panel of MMPs; except for MMP12. Transcript MMP12 was exclusively detected in HLA-G expressing JEG-3 cells but not in HLA-G deficient JAR cells. We observed HLA-G expression but no MMP12 transcription following 5-aza-2´-deoxycytidine (AZA) treatment of JAR cells. We then investigated HLA-G and MMP transcription in several human leukaemia cell lines. Leukaemia cells (lacking HLA-G expression) were converted to their HLA-G positive counterparts by AZA-treatment or by HLA-G transfection. It was found no correlation between HLA-G and MMP transcription in any examined leukaemia cell lines. The up-regulation of some MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) was observed following AZA-treatment.

Impact of blood processing on estimation of soluble HLA-G.

HLA-G is a non-classical MHC class I antigen that functions as an immunomodulatory molecule. There are two forms of HLA-G antigens, soluble and membrane bound. Soluble HLA-G can be produced by translation of HLA-G transcripts (HLA-G5, -G6, -G7) and by shedding/proteolytic cleavage of membrane bound antigens (HLA-G1, -G2, -G3, -G4). Soluble as well as membrane bound HLA-G molecules have a direct inhibitory effect on immune responses. The relevance of soluble HLA-G in various pathologic conditions, such as transplantation, autoimmunity, infectious and malignant diseases, has been extensively investigated, however interpretation remains controversial. In this work we analyzed the levels of sHLA-G (sHLA-G1 and HLA-G5) in different blood samples of healthy donors as serum, and blood plasma isolated using anti-coagulant EDTA and heparin, respectively. We found that the levels of sHLA-G (sHLA-G1 and HLA-G5) in blood plasma prepared with EDTA were significantly higher than those observed in plasma with heparin or in serum. Finally we detected the average levels of sHLA-G in females exceeded those of males.

Activation of HLA-G expression by 5-aza-2 - deoxycytidine in malignant hematopoetic cells isolated from leukemia patients.

Human leukocyte antigen - G (HLA-G) is a non-classical HLA class I antigen with restricted distribution in normal tissues. Ectopic HLA-G expression observed at some pathological circumstances as malignant transformation might be triggered by epigenetic modifications such as DNA demethylation. Recently it was demonstrated that DNA methyltransferase inhibitor 5-aza-2 - deoxycytidine (AdC) induces/enhances HLA-G transcription in many leukemia cell lines of different origin. Here we investigated the effect of AdC on HLA-G expression in malignant hematopoetic cells isolated from patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (B-CLL). We detected HLA-G expression in untreated cells from some patients. Nevertheless treatment with 5-aza-2 - deoxycytidine enhanced HLA-G transcription and concomitantly HLA-G protein synthesis in some leukemia cells.

Demethylating agent 5-aza-2'-deoxycytidine activates HLA-G expression in human leukemia cell lines.

HLA-G is a non-classical HLA class I antigen primarily expressed in the extravillous cytotrophoblast. HLA-G can also be expressed at some pathological circumstances and may thus contribute to inhibition of efficient immune responses. Complex regulation of HLA-G expression also involves epigenetic mechanisms as DNA methylation. Here we demonstrate that treatment with demethylating agent 5-aza-2'-deoxycytidine (AdC) resulted in HLA-G transcription in 18 out of 20 examined leukemia cell lines. HLA-G protein synthesis was detected in 10 cell lines expressing significant level of HLA-G transcripts following AdC treatment.

Modulation of HLA-G expression.

Recent studies demonstrated that HLA-G transcription is in some cells silenced by epigenetic mechanisms as DNA methylation and histone modification. Accordingly HLA-G gene transcriptions can be activated in such cells by demethylating agent or by inhibitors of histone deacetylation. In addition to epigenetic alterations HLA-G gene transcription can be activated by stress. In the present study these aspects of HLA-G expression are re-examined and a new inhibitor of histone deacetylation (valproic acid) and hypoxia mimetic chemical (CoCl2) are included. The highest activation of HLA-G transcription was achieved by treatment of choriocarcinoma JAR and lymphoblastoid RAJI cell lines with demethylating agent 5-aza-2 - deoxycytidine. Treatment of JAR and RAJI cells with histone deacetylase inhibitors (sodium butyrate and valproic acid) also enhanced HLA-G transcription. Nevertheless this increase in HLA-G expression was low as compared with activation by 5-aza-2 - deoxycytidine. The hypoxia mimetic agents (desferrioxamine or CoCl2) had no detectable effect on HLA-G gene transcription in examined cells. Relatively high increase of HLA-G transcription was detected in JAR and RAJI cells exposed to heat shock treatment. Interestingly heat shock induced high expression of HLA-G6 transcript in JAR cells. Heat shock treatment had no effect on alternative splicing of constitutively expressed HLA-G mRNA in choriocarcinoma cell line JEG-3. HLA-G1 protein expression was induced in JAR and RAJI cell lines by 5-aza-2 - deoxycytidine. In agreement with the differences in the levels of HLA-G transcripts JAR cells express more of HLA-G1 protein than RAJI cells.

Effect of proteasome inhibitors on expression of HLA-G isoforms.

HLA-G primary transcript is alternatively spliced into a number of mRNAs. In addition to full length HLA-G1 protein isoform these mRNAs might also encode truncated HLA-G protein isoforms lacking one or two extracellular domains. Whereas HLA-G1 protein isoform is regularly identified, truncated HLAG protein isoforms are not detected even if all alternative spliced mRNAs are present in cells. The absence of entire domain(s) renders the truncated HLA-G protein isoforms incapable of binding peptide and beta2-microglobulin. These features of truncated HLA-G protein isoforms may result in their rapid degradation by proteasomes. Here we show that despite the presence of all alternatively spliced HLA-G transcripts in JEG-3 cells pretreated with proteasome inhibitors only a full length HLA-G1 protein isoform was regularly detected. Interestingly, immunoblot analysis showed slight increase of HLA-G1 protein in cells pretreated with proteasome inhibitors, although the expression of HLA-G1 transcript was basically not affected. Expression of HLA-G3 transcript increased in JEG-3 cells pre-incubated with LLL, however, neither HLA-G3 nor other HLA-G short protein isoform was regularly detected. In K562 transfectants proteasome inhibitor LLL greatly enhanced expression of the HLA-G1 and -G2 transcripts as well as corresponding protein isoforms. Flow cytometry analysis showed that in cells pre-treated with proteasome inhibitors cell surface expression of HLA-G1 protein decreased but the quantity of intracellularly localized HLA-G antigens increased. Altogether our results suggest that truncated HLA-G proteins isoforms are not detected in JEG-3 cells as a result of their instability and the low translation efficiency of truncated HLA-G transcripts.

Binding analysis of HLA-G specific antibodies to hematopoietic cells isolated from leukemia patients.

Expression of HLA-G on the surface of malignant hematopoietic cells isolated from leukemia patients was analyzed by flow cytometry using monoclonal antibodies (mAbs) recognizing both, intact HLA-G complex (87G, 01G and MEM-G9) as well as HLA-G free heavy chain (4H84, MEM-G/1 and MEM-G/2). Prerequisite of HLA-G detection by mAbs specific to free heavy chain was mild acid treatment, which dissociates intact HLA-G complex. All mAbs, with the exception of 4H84 mAb, did not indicate the presence of HLA-G antigen in leukemia cells. Positive staining with 4H84 mAb was detected in acid-treated cells isolated from 16 out of 30 patients. Intensity of staining increased after IFN-g pre-incubation in most cases. Immunoblot analyses and RT-PCR, however, failed to detect HLA-G antigen or HLA-G transcripts in cells that bind 4H84 mAb after acid-treatment. The binding of 4H84 mAb can be explained by the acid-induced cross-reactivity of this HLA-G specific mAb with classical HLA class I molecules [15]. The results described here further demonstrate that the HLA-G molecule is not expressed in freshly isolated human leukemia cells.

[Glutamate decarboxylase (GAD)--an autoantigen in insulin-dependent diabetes mellitus (IDDM)]. / Dekarboxyláza glutamátu (GAD)--autoantigén pri inzulín-dependentnom diabetes mellitus (IDDM).

The number of antibodies to pancreatic beta-cell antigens in IDDM increased in the last years, involving antibodies to glutamic acid decarboxylase (GADab). A short review is given about the diagnostic and prognostic value of GADab determination in IDDM. The GAD plays an important, possibly a key role in the initial immunological events leading to the destruction of beta cells. The question is open whether the immunological reaction against GAD is a primary one, or if it is a result of mimicry of a part of an infectious protein antigen (Coxackie virus). The immunological reaction to GAD is associated with both humoral and cellular responses. The cellular response seems to be more important than the humoral one. The cellular response may be mediated through the HLA complex class I cells (cytotoxic lymphocytes) and the HLA complex class II cells (helper lymphocytes). There are arguments for both possibilities. The principles of GADab determination are shortly described. (Ref. 34.)

An endogenous activator of renal glutamic acid decarboxylase effects of adenosine triphosphate, phosphate and chloride on the activity of this enzyme.

The renal glutamic acid decarboxylase (GAD) differs from the brain and pancreatic enzyme by its strong binding to membranes that is not influenced by detergents. After centrifugation of freshly prepared homogenate of the rat renal cortex, only 10-15% of GAD activity was found in supernatants and 15-30% in pellets. The majority of the GAD activity was lost. The bound GAD was found in the pellet. A thermolabile activator was present in the supernatant, which was not lost on dialysis. Approximately 55% of the total GAD activity was solubilized in homogenates stored for 24 h at 4 degrees C without detergent, whereas in homogenates stored with Triton X-100, the solubilized GAD increased to 80%. This solubilization was decreased by inhibitors of thioproteases such as leupeptin, antipain and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64). Solubilized GAD was applied to DEAE Toyopearl resin and the GAD activator was eluted with 35 mM Pi. GAD was eluted with 250 mM Pi. The effect of ATP on the activity of renal GAD was also different to its effect on brain GAD. ATP is a strong inhibitor of the brain enzyme at physiological concentrations. ATP (and Pi), together with chlorides (another brain GAD inhibitor), stabilize the renal GAD. However, renal GAD was inhibited by ATP in the presence of leupeptin in freshly prepared homogenates. Similarly, ATP inhibits solubilized GAD from homogenates stored without Triton X-100 for 24 h at 4 degrees C, but Pi retains its stabilizing effect in this preparation. A significant finding of the work presented here is the obligatory requirement of an endogenous activator for renal GAD activity. Whether this activator is an enzyme converting the inactive GAD to active enzyme (as hypothesized for brain GAD), or whether it is a protein affecting the activity of renal GAD by binding (as observed for GAD in some plants) remains to be established.