[Molecular mechanisms of impaired antigenic presentation as a cause of tumor escape from immune surveillance]. / Molekulyarnye mekhanizmy narusheniya prezentatsii antigena kak prichiny uskol'zaniya opukholi ot deistviya immunnogo nadzora.

The review summarizes data on the features of antigen presentation in tumor cells. The molecular mechanisms of the antitumor immune response are considered with an emphasis on the ability of tumor cells to avoid the action of immune surveillance. The features of expression of MHC molecules depending on treatment regimens are provided. Ways to improve existing and create new treatment regimens aimed at elimination of tumor cells because of antitumor immune response are discussed.

Tumor-Infiltrating Lymphocytes in Breast Cancer. Association with Clinical and Pathological Parameters.

In patients with primary resectable breast cancer, a positive correlation between the age and the count of CD16+ lymphocytes and a negative correlation of this parameter with the number of regulatory CD4+CD25+CD127- cells and proliferative activity of Ki-67 tumor cells were revealed. Higher level of Ki-67 was associated with reduced number of effector lymphocytes (CD8+ and CD16+) and elevated content of regulatory CD8+CD11b-CD28- T cells. The absence of expression of estrogen receptors was associated with reduced cytotoxic potential of CD8+ T cell in comparison with ER+ breast cancer. The percentage of CD8+ lymphocytes (CD3+CD8+ and CD8+CD11b+CD28+) among lymphocytes infiltrating the tumor was higher in PR+ breast cancer than in PR- tumors. With increasing the tumor load, the number of lymphocytes expressing CD16 marker and their cytotoxic potential decreased.

Comparative analysis of cells with combined apoptosis and proliferation markers in thyroid tissue specimens from patients with cancer, adenoma, and autoimmune diseases.

Combined phenotypes of cells with membrane and intracellular expression of apoptosis and proliferation regulation markers (p53, bcl-2, CD95, CD95L, Ki-67) were studied by flow cytometry of cell suspension from thyroid tissue specimens from patients with autoimmune diseases, adenoma, and thyroid cancer. The incidence of cell groups with phenotypes p53/Ki-67, p53/CD95, bcl-2/Ki-67, bcl-2/CD95, CD95/Ki-67, p53/CD95L, CD95/CD95L, and bcl-2/CD95L was evaluated and the density of receptor distribution on/in each cell group are presented. Patients with autoimmune diseases had high incidence of cells with phenotypes p53/Ki-67, p53/CD95, bcl-2/Ki-67, bcl-2/CD95, CD95/Ki-67, p53/CD95L, CD95/CD95L, and bcl-2/CD95L; cells with the bcl-2/CD95 phenotype were the most incident. Patients with thyroid adenoma had high levels of cells with p53/CD95L phenotype, while patients with thyroid cancer had significantly lower levels of p53 expression in the p53/CD95L cell group. The density of CD95L receptors on CD95/CD95L-positive cells was 4-7-fold higher in patients with thyroid tumors; the density of CD95L receptors on CD95/CD95L cells was maximum in thyroid adenoma and minimum in thyroid cancer. These data indicate differences in the expression of apoptosis and proliferation markers in thyroid adenoma, cancer, and autoimmune diseases. Analysis of the expression of these markers in the above diseases can be useful for differential diagnosis.

[Hydrophilic regulator hexapeptides as inhibitors of tumor cell multiple drug resistance].

Multiple drug resistance (MDR) of tumor cells to cytostatics is one of the most often and severe complications of chemotherapy in oncological patients. The phenomenon of MDR could be due to a sharp increase of the activity of the ATP-dependent transport proteins of the ABC system, that provides pumping of the drug from the cells to the extracellular space. Up to now, all the attempts to design agents preventing MDR were of no success. One of the prospective trends is the use of hydrophilic regulator hexapeptides. Three regulator hydrophilic hexapeptides of the linear and cyclic structure were used as the MDR modulators. The sensitivity of the tumor cells to various cytostatics in the presence of the peptides was determined by the MTT-test and the direct counting of the survived cells. The effect of the hexapeptides on MCF7, KB8-5 and PC3 cells was investigated. It was concluded that the hydrophilic hexapeptides of the linear and cyclic structure increased the sensitivity to doxorubicin (a cytostatic). The tumor cell MDR inhibition was mediated by the ATP-dependent transport protein MRP. Such a characteristic of the hexapeptides is of interest for their use as agents preventing MDR.

Role of P-glycoprotein in evolution of populations of chronic myeloid leukemia cells treated with imatinib.

Imatinib mesylate (imatinib) is a new generation preparation that is now successfully used for treatment of cancer, particularly for chemotherapy of chronic myeloid leukemia (CML). Imatinib inhibits the activity of chimeric kinase BCR-ABL, which is responsible for the development of CML. The goal of this study was to investigate the role of a multidrug resistance protein, P-glycoprotein (Pgp), in the evolution of CML treated with imatinib. We demonstrate here that although imatinib is a substrate for Pgp, cultured CML cells (strain K562/i-S9), overexpressing active Pgp, do not exhibit imatinib resistance. Studies of CML patients in the accelerated phase have shown variations in the number of Pgp-positive cells (Pgp+) among individual patients treated with imatinib. During treatment of patients with imatinib for 6-12 months, the number of Pgp-positive cells significantly increased in most patients. The high number of Pgp+ cells remained in patients at least for 4.5 years and correlated with active Rhodamine 123 (Rh123) efflux. Such correlation was not found in the group of imatinib-resistant patients examined 35-60 months after onset of imatinib therapy: cells from the imatinib-resistant patients exhibited efficient Rh123 efflux irrespectively of Pgp expression. We also compared the mode of Rh123 efflux by cells from CML patients who underwent imatinib treatment for 6-24 months and the responsiveness of patients to this therapy. There were significant differences in survival of patients depending on the absence or the presence of Rh123 efflux. In addition to Pgp, patients' cells expressed other transport proteins of the ABC family. Our data suggest that treatment with imatinib causes selection of leukemic stem cells characterized by expression of Pgp and other ABC transporters.

Variations in the number of regulatory T cells (CD4+CD25+) in patients with breast cancer during herceptin therapy.

We studied the effect of Herceptin therapy on the population composition of lymphocytes and percentage of CD4+CD25+ cells (regulatory T cells) in breast cancer patients. Herceptin treatment decreased the number of "professional" T suppressors (CD4+CD25+ cells and regulatory T cells) in the peripheral blood.

Intracellular localization and content of YB-1 protein in multidrug resistant tumor cells.

The multifunctional mammalian protein YB-1 is a member of the large DNA- and RNA-binding protein family with an evolutionarily ancient cold-shock domain. YB-1 is involved in multiple DNA- and mRNA-dependent events and regulates gene expression at various levels. It can be found both in the nucleus and the cytoplasm. Bound to DNA in the cell nucleus, YB-1 functions as a transcription factor interacting with inverted CCAAT-box (Y-box) in promoters and enhancers of multiple genes. In particular, YB-1 regulates activity of the multidrug resistance (MDR) genes MDR1 and LRP. In tumors, YB-1 has been suggested to be an early and global marker of MDR. In this study, we compared amounts of YB-1 mRNAs and intracellular localization of YB-1 protein in six pairs of drug sensitive and drug resistant sublines of diverse tumors. We have shown that neither great increase in the level of YB-1 mRNA nor substantial increase in the number of cells with nuclear localization of YB-1 are obligatory traits of drug resistant tumor cell populations. However, the cells with highest amounts of YB-1 mRNA also demonstrated increased quantities of MDR1, MRP1, BCRP, and LRP mRNAs encoding different MDR proteins. Transfection of two different populations of drug-sensitive cells with YB-1 cDNA led to increase in the amount of YB-1 mRNA. The quantities of MRP1 and LRP mRNAs increased in both populations. Introduction of YB-1 small hairpin RNA (shRNA) resulted in decreased amounts of YB-1 mRNA, as well as MRP1, LRP, and MDR1 mRNAs (in three different cell lines). Our data suggest that although YB-1 regulates several MDR genes, it could not be regarded as a global marker of already formed drug resistant tumor cell populations. It is most likely that at the first steps of MDR development YB-1 activity is necessary for propagation of resistant cell populations rather than for maintenance of drug resistance.

New approach to vital analysis of functional activity in ABC transporters (markers for multidrug resistance) in solid tumors by the method of flow cytofluorometry.

We developed and described a new approach to vital analysis of functional activity of multidrug resistance markers (ABC transporters) in intact biopsy specimens from human solid tumors by the method of flow cytofluorometry. The algorithm of the study underwent revision, and the cell suspension was obtained in the final stage. Intensification of intracellular doxorubicin accumulation (fluorescence) and increase in the number of fluorescent cells and total fluorescence of doxorubicin-accumulating cells produced by ABC transporter inhibitor sodium azide served as the criteria of expression of these transporters in tumor tissue. Informative value of changes in various parameters of doxorubicin fluorescence is discussed. The increase in the count of fluorescent cells in the suspension of tumors cells after treatment with the inhibitor indicates the presence of tumor cells absolutely resistant to this preparation. The proposed method is technically simple, suitable for structurally different tumors, requires small amounts of the biopsy material and, therefore, can be used for routine analysis. The results of our analysis and spectrofluorometric assay of ABC transporters agree very closely, which suggest that this method is adequate for the purpose.

[Inhibition of ABC-transporter(s)' function in non-small cell lung cancer cells by platinum drugs]. / Ingibirovanie funktsii ABC-transporterov v kletkakh nemelkokletochnogo raka legkogo pri vozdeistvii preparatov platiny.

With an account of the literature data that platinum drugs react with many cellular targets, including ATP and proteins, the authors suggested that disturbance of the function of energy-dependent ABC-transporters (markers of multidrug resistance, MDR) under the effect of platinum drugs could be a cause of increased efficacy of MDR agents (agents, MDR to which is developed by the classical mechanism) when used in combination with platinum drugs even in the treatment of multidrug resistant lung cancer. The cisplatin and carboplatin effect on accumulation of MDR doxorubicin in cells of non-small cell cancer was studied by flow cytometry with the use of biopsy specimens. The MDR phenotype of the tumors was determined by a change in doxorubicin intracellular accumulation under the action of the ABC-transporter(s)' inhibitors: verapamil and genistein (specific inhibitors of Pgp and MRP respectively) and sodium azide (an inhibitor of all energy-dependent ABC-transporters). The MDR phenotypes, i.e. Pgp-MRP+ or Pgp+MRP+, were detected in all the tumors investigated. Two types of changes in doxorubicin intracellular accumulation under the action of the inhibitors and the platinum drugs were shown: (a) an increase in doxorubicin cytoplasmic accumulation and (b) a change in subcellular distribution of the anthracycline (increased accumulation of doxorubicin in the cell nucleus and its higher binding to DNA). Cisplatin and carboplatin had an inhibitory effect on ABC-transporter(s) in all the tumors investigated but the effect of carboplatin was less pronounced. It was concluded that cisplatin and carboplatin stimulation of doxorubicin intracellular accumulation, as well as a change in subcellular distribution of the anthracycline under the action of the platinum drugs (increased doxorubicin accumulation in the cell nucleus) in multidrug resistant lung tumors could be at least partly explained by inhibition of the MDR transporter(s)' function. The results could provide a basis for the use of the sequential combination cisplatin (or carboplatin)-->doxorubicin in the treatment of multidrug resistant lung cancer.

[Relationship between the induction of MDR1, a multidrug resistance gene in tumor cells, and apoptosis]. / Sviaz' mezhdu inductsiei aktivnosti gena mnozhestvennoi lekarstvennoi ustoichivosti opukholevykh kletok MDR1 i apoptozom.

Gene MDR1 coding for P-glycoprotein belongs to a group of genes responsible for cell defense. Overexpression of this gene determines the resistance of tumor cells to a series of chemotherapeutic drugs known as multidrug resistance. Many chemotherapeuticals induce both apoptosis and transcriptional activity of the MDR1 gene in tumor cells. It is not known, however, how these two processes are associated with each other. In order to elucidate a possible link between them, we have studied the sphyngomyelinic pathway of signal transduction. This pathway is activated in response to various stress factors and includes the hydrolysis of sphyngomyelin of cytoplasmic membrane resulting in an accumulation of intracellular ceramide, which activates cascades of enzymatic reactions leading to various cell responses, including apoptosis. C2 ceramide (N-acetyl-D-sphyngosine) and cytosar (1 beta-D-arabinosylcytosine, or ara C) were used to induce the sphyngomyelinic pathway. Their effects on human hemoblastosis cell lines (K562 and H9 cell lines) were examined. C2 ceramide and ara C induced apoptosis in both cell lines over an 18-h incubation. C2 ceramide also induced an increase in the expression of the gene MDR1 in both cell lines, while ara C increased the activity of the gene MDR1 only in H9 cells. The results obtained provide evidence for the contribution of ceramide-mediated signal pathway to the control of MDR1 activity.

[Functional activity and expression of P-glycoprotein in chronic myeloid leukemia]. / Funktsional'naia aktivnost' i ékspressiia P-glikoproteina pri khronicheskom mieloleikoze.

AIM: To evaluate the prognostic significance of P-glycoprotein (Pgp) in chronic myeloid leukemia (CML). MATERIALS AND METHODS: Functional activity (rhodamine 123 test) and expression of Pgp (binding of UIC2 monoclonal antibodies by cells) were evaluated by flow cytofluorometry. A total of 141 samples of peripheral blood from 121 patients with various stages of CML were examined. RESULTS: The number of patients whose cells express functionally active Pgp increases during the blast crisis (BC) in comparison with the chronic phase (CP). Repeated testing of patients with BC and CP showed that Pgp-expressing cells can disappear from the peripheral blood of patients despite the treatment by Pgp preparations and substrates. However the number of cases with expression and functional activity of Pgp increases in the course of BC. Several patients in whom functionally active Pgp was not detected during diagnosis of BC had longer BC phase than patients with the active protein. CONCLUSION: These data suggest that active Pgp contributes to CML BC (presumably to patient's response to therapy) but this contribution is not decisive.

[Characteristics of drug resistance of tumor plasmocytes in vitro in patients with multiple myeloma differently responsive to chemotherapy]. / Kharakteristika lekarstvennoi ustoichivosti opukholevykh plazmotsitov in vitro u bol'nykh mnozhestvennoi mielomoi s razlichnym otvetom na khimioterapiiu.

AIM: To determine sensitivity of tumor plasmocytes in vitro to cytostatic drugs (prednisolone, alkeran belustin, vincristine, rubomycin, doxorubicin, cytarabin, methotrexate, cysplatin, etoposide). MATERIAL AND METHODS: The sensitivity was measured with DISC method in 12 patients with multiple myeloma (MM) in two groups: resistant and responsive to induction polychemotherapy (PCT). RESULTS: The groups appeared significantly different by lowering of pathological paraprotein concentration (PIg): by 7.4 +/- 2.5% and 32.5 +/- 3.7%, respectively (p < 0.05). The resistance to the drugs was higher in the resistant patients than in the responders (0.7 +/- 0.28 versus 0.4 +/- 0.02, p < 0.05). PCT schemes of resistant patients contained 65.0 +/- 2.3% of ineffective drugs. In the responders the percentage was 35.7 +/- 5.3% (p < 0.05). CONCLUSION: The relationship exists between resistance of tumor plasmocytes to drugs in vitro and clinical findings.

CD95 (FAS/APO-1) antigen is a new prognostic marker of blast cells of acute lymphoblastic leukaemia patients.

We analyzed CD95(Fas/APO-1) antigen expression on bone marrow blasts in 38 children with acute lymphoblastic leukemia (ALL) receiving a treatment in the Department of Leukaemias at the Cancer Research Center in 1987-1989 years (n = 22) and in 1994-1997 years (n = 16). CD95 antigen expression was studied by monoclonal antibodies (MoAbs) IPO-4 in indirect immunofluorescence analysis. CD95 antigen was expressed on 35.8 +/- 7.5% bone marrow blasts, most frequently (63.6%) in the clinically favourable Pre-B ALL. Only in this group CD95 antigen expression was correlated with CD10 antigen expression that has a positive influence to the time of complete remission in ALL patients. Our data showed that CD95 expression on blast cells is a favourable prognostic sign, associated with increased relapse-free and total survival. On the contrary, the absence of CD95 antigen on blasts is an unfavourable sign for disease evolution.

Studies of some mechanisms of drug resistance in chronic myeloid leukemia (CML).

CML is the myeloproliferative disorder connected with the specific chromosome translocation (9;22) and occurrence of the fusion gene/protein BCR-ABL. BCR-ABL protein is believed to inhibit apoptosis and to cause drug resistance. We investigated the correlation of two different forms of BCR-ABL mRNA in 94 pts with their overall survival. It was found that b2a2 (but not b2a3) mRNA expression correlates with longer survival of patients treated with chemotherapy. We did not find an influence of different types of BCR/ABL mRNA on the survival of pts treated with interferon-alpha. FAS/APO-1 antigen was expressed by the cells of 34% of the pts in CML blast crisis (BC) and directly correlated with the the expression of CD34, CD13 and CD14 differentiation antigens. FAS/APO-1 non-expression correlated with higher rate of remissions in BC. We investigated P-glyco-protein (Pgp) expression and functional activity in 40 BC CML pts. 2-fold shorter survival was found in the pts with Pgp expression. Pgp expression strongly correlated with CD13 antigen. Consecutive studies of pts in BC CML show that Pgp expressing cells often do not multiply in the course of BC CML. We postulate that Pgp may be regarded as differentiation marker of the cells and the unfavorable prognostic factor in BC CML.

[An evaluation of the prognostic significance of antigen CD95(Fas/APO-1) expression on the cells of patients with a myelodysplastic syndrome, acute myeloid leukemia and chronic myeloleukemia]. / Otsenka prognosticheskoi znachimosti ékspressii antigena CD95(Fas/APO-1) na kletkakh bol'nykh s mielodisplasticheskim sindromom, ostrym mieloidnym leikozom i khronicheskim mieloleikozom.

AIM: The expression of CD95(Fas/APO-1) antigen was studied on bone marrow cells of 19 MDS patients, peripheral blood blast cells of 15 acute myeloid leukemia (AML) patients, blast cells and granulocytes of 68 patients with chronic myeloid leukemia (CML)--24 in chronic, 9 in accelerated phase and 35 in blastic crisis (BC)--by indirect surface immunofluorescence assay using flow cytometry (FACScan, Becton Dickinson, USA). RESULTS: CD95(Fas/APO-1) antigen was revealed on bone marrow cells of 8 out of 19 (36.8%) MDS patients; the percentage of antigen-positive cells was 38.1 +/- 19.2%; on 45.5 +/- 22.8% of cells in 6(45%) of 15 AML patients. Fas/APO-1 antigen was totally absent in CML chronic stage; its expression was found in 34% (12 of 35) of our patients with CML BC on peripheral blood blasts and in 56% (5 of 9) on peripheral blast cells of CML patients in acceleration phase. CONCLUSION: The data on overall survival of CD95-positive MDS patients suggest that the presence of Fas antigen is a favorable prognostic sign for patients with MDS. The patients from CD95-negative group represent a risk group both for survival and AML transformation. In CML BC group the survival does not depend upon Fas-antigen expression.

Influence of exogenous RAR alpha gene on MDR1 expression and P-glycoprotein function in human and rodent cell lines.

The goal of our study was to obtain direct evidence of co-ordinated regulation of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) and differentiation in tumour cells and to study some signalling pathways involved in joint regulation of these two cell phenotypes. The sublines of human melanoma (mS) and hepatoma (human HepG2 and rat McA RH 7777) cell lines were obtained by retroviral infection of the wild-type cells with the cDNA of the human retinoic acid receptor alpha (RAR alpha). The resulting sublines stably overexpressed exogenous RAR alpha gene. The infectants became more differentiated than the parental cells as determined by a decrease in the synthesis of the embryo-specific alpha-fetoprotein in HepG2 and McA RH 7777 hepatoma cells and by an increase in melanin synthesis in mS cells. The differentiation of human cells was accompanied by an increase in the amounts of MDR1 mRNA but not by an increase in P-gp activity as a drug transporter, in contrast, in the rat RAR alpha overexpressing cells P-gp functional activity was elevated. Treatment with cytotoxic drug (colchicine) or retinoic acid (RA) resulted in a slight increase in P-gp activity in the parental and RAR alpha-infected melanoma cells, whereas the increase in P-gp function in the infected hepatoma cells (both human and rat) was very prominent. Thus, we provide new evidence that cell differentiation caused by the overexpression of the gene participating in the differentiation programme leads to overexpression of MDR1 gene and drug resistance and that this effect is tissue and species specific. These data imply that the activation of the RA-controlled signalling pathway up-regulates MDR1 gene expression.

Coordinated regulation of P-glycoprotein activity and cytochrome P-4501A induction in sublines of rat hepatoma McA RH7777 cells with different levels of colchicine resistance.

The constitutive and induced activities of cytochrome P-4501A isoforms in hepatoma McA 7777 sublines with different levels of colchicine (CH) resistance were studied. The higher CH resistance was associated with the elevated functional activity of P-glycoprotein (Pgp). The constitutive level of benzo(a)pyrene hydroxylase and 7-ethoxyresorufin O-deethylase (cytochrome P-4501A-dependent activities) were the same in sublines with different CH resistance levels. However, benzo(a)-anthracene, a cytochrome P-4501A inducing agent, more effectively induced benzo(a)pyrene hydroxylase and 7-ethoxyresorufin O-deethylase activities in sublines with elevated P-glycoprotein activity. The toxicity of benzo(a)pyrene, a compound which is simultaneously a cytochrome P-4501A-inducing agent and a toxic agent activated by cytochrome P-4501A, is more effective in sublines with elevated CH resistance. These results support the suggestion about the coordinated regulation of enzyme systems involved in the defence against various lipophilic xenobiotics. The possibility to overcome the Pgp-mediated MDR of some tumours by using a combination of some drugs including compounds which induce the cytochrome P-4501A isoforms and are activated by them is discussed.

Fas (APO-1/CD95) Antigen: New Activation Marker for Evaluation of the Immune Status.

Using mAb ICO-160, we have studied Fas (APO-1/CD95) antigen expression on blood lymphocytes from healthy women, pregnant women and patients with chronic adnexitis, uterin myoma, ovarian cyst, ovarian and uterin cancer. In peripheral blood from healthy women Fas antigen was detected on 23.42 +/- 2.9% of lymphocytes. The healthy donors were divided in two different subgroups with low and high Fas expression. Expression of Fas antigen on lymphocytes was elevated (high expression subgroup) in all the groups of investigated patients, excepting the ovarian cancer ones with Fas expression similar to that in healthy donors. Comparison of expression of other differentiation antigens between healthy donors and the above groups of patients elucidated in the patients a kind of pronounced imbalance of the immune status and activation of the immune system. Additionally, in patients with ovarian cancer the imbalance of the immune status was reflected as altered ratio between helper and suppressor cells (the ratio was 0.73 in contrast to that 1.08 in group of healthy donors). As follows from the summarized results of all the trials, Fas antigen expression correlated with expression of other activation antigens as CD71 and CD25 (r = 0.05 and r = 0.52, respectively). Consequently, Fas (APO-1/CD95) antigen may be considered as the activation antigen and its expression may be used for assessing the immune status.