Par6G suppresses cell proliferation and is targeted by loss-of-function mutations in multiple cancers.

Differentiated epithelial structure communicates with individual constituent epithelial cells to suppress their proliferation activity. However, the pathways linking epithelial structure to cessation of the cell proliferation machinery or to unscheduled proliferation in the context of tumorigenesis are not well defined. Here we demonstrate the strong impact of compromised epithelial integrity on normal and oncogenic Myc-driven proliferation in three-dimensional mammary epithelial organoid culture. Systematic silencing of 34 human homologs of Drosophila genes, with previously established functions in control of epithelial integrity, demonstrates a role for human genes of apico-basal polarity, Wnt and Hippo pathways and actin dynamics in regulation of the size, integrity and cell proliferation in organoids. Perturbation of these pathways leads to diverse functional interactions with Myc: manifested as a RhoA-dependent synthetic lethality and Par6-dependent effects on the cell cycle. Furthermore, we show a role for Par6G as a negative regulator of the phosphatidylinositol 3'-kinase/phosphoinositide-dependent protein kinase 1/Akt pathway and epithelial cell proliferation and evidence for frequent inactivation of Par6G gene in epithelial cancers. The findings demonstrate that determinants of epithelial structure regulate the cell proliferation activity via conserved and cancer-relevant regulatory circuitries, which are important for epithelial cell cycle restriction and may provide new targets for therapeutic intervention.

[Effect of homologous peptides of differentiation factors HLDF and PEDF on preimplantation development of mice in vitro]. / Vliianie gomologichnykh peptidov factorov differentsirovki HLDF i PEDF na doimplantatsionnoe razvitie myshei in vitro.

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351-356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84-89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2-4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.

Effect of synthetic beta-endorphin-like peptide immunorphin on human T lymphocytes.

Beta-endorphin and the synthetic beta-endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met(5)]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of beta-endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of (125)I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (K(d) = 7.0 +/- 0.3 nM). Unlabeled immunorphin completely inhibits (125)I-labeled beta-endorphin specific binding to naloxone-insensitive receptors on T lymphocytes (K(i) = 0.6 +/- 0.1 nM). Thus, beta-endorphin and immunorphin interact with common naloxone-insensitive receptors on T lymphocytes.

[Interaction of synthetic decapeptide SLTCLVKGFY with human T lymphocytes]. / Vzaimodeistvie sinteticheskogo dekapeptida SLTCLVKGFY s T-limfotsitami cheloveka.

The synthetic peptide SLTCLVKGFY, corresponding to the 364-373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [125I] beta-endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (Ki 0.6 nM). The fragments 3-10, 4-10, 5-10, and 6-10 of Immunorphin also inhibited the binding (Ki 2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and, therefore, are not opioid. The Kd values of the specific binding of 125I-labeled Immunorphin and its 6-10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.

Synthetic beta-endorphin-like peptide immunorphin binds to non-opioid receptors for beta-endorphin on T lymphocytes.

The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [125I]beta-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K(i) = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 1.0 nM) possessed the ability to inhibit specific binding of [125I]beta-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. K(d) values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively.

[Biologically active fragment of the differentiation factor from HL-60 cell line. Identification and properties]. / Biologicheski aktivnyi fragment faktora differentsirovki kletok linii HL-60. Identifikatsiia i svoistva.

Six-membered peptide fragment TGENHR (HLDF-6) was identified in the HL-60 cell culture of human promyelocyte leukemia treated with retinoic acid when studying the differentiation factor HLDF of this cell line. HLDF-6 retains the ability of the full-size factor to induce the differentiation and arrest the proliferation of the starting HL-60 cells. It was shown that the synthetic peptide HLDF-6 has no specific receptors on the surface of the HL-60 cells but can affect the binding of interleukin IL-1 beta, a cytokine involved in proliferation, to the cell surface. It was found on a model of transplantable NSO myeloma that HLDF-6 has an antitumor activity.

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on activity of murine thymocytes and peritoneal macrophages.

The purpose of the present study was to investigate properties and mechanism of action of the synthetic adrenocorticotropin (ACTH)-like peptide VKKPGSSVKV, corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain. The ACTH-like peptide was shown to act as an immunosuppressive agent in vitro: it inhibits the blast transformation of mouse thymocytes and reduces the spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against Salmonella typhimurium 415 virulent strain bacteria. High affinity receptors for the ACTH-like peptide were found on thymocytes and macrophages and shown to be at the same time the receptors for ACTH. The kinetic characteristics of the ACTH-like peptide and 125I-labeled ACTH (13-24) (ACTH 'address segment') specific binding to the receptors were determined. It was found that the ACTH-like peptide binding to the receptors on target cells is accompanied by an increase in both adenylate cyclase activity and intracellular cAMP content.

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on proliferation of lymphoblastoid cells.

Influence of the ACTH-like peptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain on growth of MT-4 human T-lymphoblastoid cell line was investigated. It was found that the ACTH-like peptide at concentration range 10(-11) -10(-7) M inhibits the proliferation of MT-4 cells. Labeled ACTH 'address segment' [(125)I]ACTH (13-24) was used to establish that MT-4 cells express specific receptors for ACTH (K(d) = 97 pM). The ACTH-like peptide and human ACTH (but not IgG1 heavy chain) were shown to compete with [(125)I]ACTH (13-24) for binding to these receptors (K(i1) = 0.38 nM and K(i2) = 0.34 nM).

[Hormone-like activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1]. / Gormonopodobnye svoistva sinteticheskogo dekapeptida, sootvetstvuiushchego adrenokortikotropinopodobnoi posledovatel'nosti immunoglobulina G1 cheloveka.

The antiproliferative and immunosuppressive in vitro effects of immunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11-20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10(-11)-10(-7) M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strain Salmonella typhimurium 415. By using a 125I-labeled "addressing" fragment of ACTH ¿[125I]ACTH-(13-24)¿, we showed that MT-4 cells express specific receptors for ACTH (Kd 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [125I]ACTH-(13-24) to these receptors with Ki1 of 0.38 and Ki2 of 0.34 nM, respectively. Specific receptors for ACTH (Kd 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to complete with labeled ACTH-(13-24) for binding to these receptors (Ki = 1.8 nM) and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.

Study of immunosuppressive activity of a synthetic decapeptide corresponding to an ACTH-like sequence of human immunoglobulin G1.

The synthetic ACTH-like decapeptide H-Val-Lys-Lys-Pro-Gly- Ser-Ser-Val-Lys-Val-OH, corresponding to amino acid residues 11-20 of the variable part of the human IgG1 heavy chain (referred to as immunocortin) was found to have an immunosuppressive effect on cells in vitro: it inhibits blast transformation of mouse thymocytes and reduces spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against the virulent bacterial strain Salmonella typhimurium 415. Tritium-labeled immunocortin binds with high affinity to ACTH receptors on thymocytes and macrophages (Kd 2. 1 and 2.5 nM, respectively) and activates adenylate cyclase in these cells. Thus, the interaction of immunocortin with the target cell includes the following main steps: binding to the receptor, activation of adenylate cyclase, and elevation of the intracellular content of cAMP.

[Appearance of glutamate receptors on the surface of HL-60 cells upon differentiation]. / Poiavlenie glutamatnykh retseptorov na poverkhnosti kletok HL-60 pri ikh differentsirovke.

A specific interaction of L-glutamic acid with promyelocytic leukemia HL-60 cells completely differentiated by all-E-retinoic acid (Kd = 0.5 microM) and by plasma membrane fraction from the same cells (Kd = 1 microM) was detected through radioligand analysis and characterized. Quisqualate, a nonlabeled structural analogue of glutamic acid, was found to inhibit competitively the specific binding of L-[3H]glutamic acid to the membranes (Ki = 0.24 microM). The stereospecificity of the binding was demonstrated. These data suggest that specific glutamate receptors appear on the surface of HL-60 cells during their differentiation.