[Regulation of proliferation and viability of tumor cells in vitro by alloferon-1 and allostatin-1].

Alloferon-1 (AF) and allostatin-1 (AS) cytotoxic and growth modulating activities have been compared. AF is cationic oligopeptide isolated from the hemolymph of experimentally infected blow fly Calliphora vicina. AS is AF synthetic analog that differs from the parent molecule in two amino acids substituted. It has been shown that both AF and AS have no direct cytotoxic activity in concentrations ranging from 1 x 10(-1) to 10 microg/ml, however, the peptides demonstrated significant effect on tumor cells proliferation in vitro. Both peptides displayed growth modulating activity in mass cell cultures and boosted growth inhibiting activity of doxorubicin in the course of P388D1 cells cloning, although AS potentated doxorubicin cytostatic activity to a greater extent. Similarly, AS boosted anti-clonogenic activity of cyclophosphamide applied in a subthreshold concentration. Experiments with peptide-fluorescein complex have demonstrated that AF and AS belong to the group of cell-penetrating peptides. Moreover, the experiments displayed AF ability to bind with chromosomes.

[Growth modulating and cytotoxic effects of the peptide from hemolymph of blow fly Calliphora vicina (Diptera, Calliphoridae) in vitro].

Biological activity of alloferon (AF), peptide, consisting of 13 a. a. isolated from hemolymph of experimentally infected blow fly Calliphora vicina was studied. AF in concentrations form 1 x 10(-5) to 250 microg/ml was added into the culture medium of the target cell lines K562, J-96, P388DI, Hep22a and 3T3B-SV40. First two days the peptide in concentrations 1 x 10(-5) and 1 x 10(-3) microg/ml in most cases stimulated the cell proliferative activity and suppressed the cell growth when applied in concentrations 10 and 100 microg/ml. Trend in growth modulating effect was dependent on duration of AF treatment. The peptide did not expressed cytotoxic effect with the exception of destruction of P388D1 cells that was registered after 96 h incubation in the medium initially contained 100 microg/ml AF. Simultaneously, cytotoxic and growth modulating effects of doxorubicin and cytosinarabinoside, as well as hybrid molecules, AF--cytosinarabinoside (cytal) and AF--doxorubicin (doxal) have been studied. Doxorubicin and cytosinarabinoside expressed greater growth inhibition effect compared to the hybrid molecules and AF itself. The results obtained with mass cell cultures were supported by experiments where P388D1 cells clonogenic capacity was tested. Besides, it has been demonstrated that AF rapidly penetrates into cytoplasm of J-96 cells and concentrates mainly into and around the cell nuclei.

Antiviral and antitumor peptides from insects.

Insects can rapidly clear microbial infections by producing a variety of immune-induced molecules including antibacterial and/or antifungal peptides/polypeptides. In this report, we present the isolation, structural characterization, and biological properties of two variants of a group of bioactive, slightly cationic peptides, referred to as alloferons. Two peptides were isolated from the blood of an experimentally infected insect, the blow fly Calliphora vicina (Diptera), with the following amino acid sequences: HGVSGHGQHGVHG (alloferon 1) and GVSGHGQHGVHG (alloferon 2). Although these peptides have no clear homologies with known immune response modifiers, protein database searches established some structural similarities with proteins containing amino acid stretches similar to alloferon. In vitro experiments reveal that the synthetic version of alloferon has stimulatory activities on natural killer lymphocytes, whereas in vivo trials indicate induction of IFN production in mice after treatments with synthetic alloferon. Additional in vivo experiments in mice indicate that alloferon has antiviral and antitumoral capabilities. Taken together, these results suggest that this peptide, which has immunomodulatory properties, may have therapeutic capacities. The fact that insects may produce cytokine-like materials modulating basic mechanisms for human immunity suggests a source of anti-infection and antitumoral biopharmaceuticals.

[Cytotoxic and mitogenic effect of antimicrobial peptides from neutrophils on cultured cells]. / Tsitotoksicheskoe i mitogennoe vliianie antimikrobnykh peptidov neitrofilov na kul'tiviruemye kletki.

Cytotoxic and mitogenic activities of human and rabbit defensines (HNP and NP-2, resp.) and pig antimicrobial peptides from leukocytes (PR-39, prophenin PF-2 and protegrin PG-2) were studied. The above peptides were added to serum-free cell culture medium of the target cell lines K562, L929 and Hep22a. Cytotoxicity was estimated within 1, 3, 6, 24 and 48 h of cell incubation with the tested peptides in concentrations 1, 10, 25 or 100 micrograms/ml. All the examined peptides exhibited a distinct time- and concentration-dependent cytotoxicity. Moreover, by contrast to pig peptides, defensines could induce proliferation in cell subpopulations from cell lines L929 amd Hep22a, or L929 (defensines HNP and NP-2, resp.), keeping resistance to their cytotoxic action.

[The release and absorption of small ribonucleoprotein complexes (alpha RNPs) in cultures of transformed rat fibroblasts and human epidermoid carcinoma]. / Eksport i pogloshchenie malykh ribonukleoproteinovykh kompleksov (al'fa RNP) v kul'turakh transformirovannykh krysinykh fibroblastov i épidermoidnoi kartsinomy cheloveka.

The release of alpha RNPs and their absorbtion by the cells from culture medium were studied. The rat fibroblasts of parental serum-dependent cell line LRec-1, and of selected serum-free cell line LRec-1sf rapidly released and absorbed alpha RNPs. In the latter case, both auto- and heterologous alpha RNPs were seen to penetrate, whereas only autologous alpha RNPs entered LRec-1 cells. Besides, the ability to rapid export and absorbtion of autocrine alpha RNPs was demonstrated for human epidermoid carcinoma cell line A431. Both LRec-1 and LRec-1sf cells expressed mRNAs with homology to ID-like nucleotide sequences, the level of mRNA expression decreasing markedly when serum-dependent LRec-1 cells were grown in serum-free medium.

[The immunomodulating and immunoprotective activity of small ribonucleoprotein complexes (alpha RNPs) from the LRec-1sf cell line]. / Immunomoduliruiushchaia i immunoprotektivnaia aktivnost' malykh ribonukleoproteinovykh kompleksov (al'fa RNP) iz kletok linii LRec-1sf.

Effect of nuclear and released into culture medium alpha RNPs (N- and R-alpha-RNPs, resp.) produced by transformed rat embryo fibroblasts of serum-free cell line LRec-1sf on the nonsensibilized mouse splenocyte cytotoxicity (NK-mediated cell lysis) was studied. A preliminary treatment with N-alpha-RNPs resulted in decreasing K562 cell sensitivity to splenocyte cytotoxicity, whereas pretreatment of the splenocytes themselves exerted no cytotoxic effect. The target cell preincubation with R-alpha-RNPs had no influence on K562 cell resistance to NK cell cytotoxicity. The identical splenocyte preincubation was without action on their cytotoxic effect to LRec-1sf cells, however, resulted in an increase of the K562 cell lysis. The addition of R-alpha-RNPs into splenocyte/target cell mixtures had no influence on NK-mediated lysis, when K562 cells were used as a target cell line, but suppressed the NK-mediated lysis of LRec-1sf cells. The results of the present experiments suggest that alpha RNPs produced by LRec-1sf cell line exhibit the capacity for modulating both mouse NK cytotoxicity, and the transformed cell sensitivity to NK-mediated lysis.

[The growth-stimulating and immunomodulating activity of culture media conditioned by transformed fibroblasts from serum-free lines]. / Roststimuliruiushchaia i immunomoduliruiushchaia aktivnost' kul'tural'nykh sred, konditsionirovannykh transformirovannymi fibroblastami bessyvorotochnykh linii.

Five cell lines of transformed rodent fibroblasts capable of unlimited growth without exogenous proteins were studied. It has been demonstrated that the media conditioned by these cells (CMs), the protein preparations obtained from CMs, or the feeder cell cultures of the serum-free cell lines are mitogenic to mouse hybridoma cells as well as to primary cell cultures of the rat bone marrow. The addition of CMs proteins into mixtures of mouse splenocytes and transformed target cells resulted in stimulation or suppression of the splenocyte cytotoxicity when YAC-1 [correction of JAC-1] or K562 cell targets were used. A 18-20 h splenocyte preincubation with proteins, released by transformed rat fibroblasts of the serum-free cell line LRec-1sf, resulted in a 1.5-4-fold increase in K562 cell lysis by murine natural killer cells. By contrast, the target cell preincubation with the same proteins resulted in a 1.5-2-fold decrease in K562 cell sensitivity to cytotoxic effect of natural killer cells. Taken together our data show that the transformed rodent fibroblasts, growing without exogenous proteins, produce and release into the culture medium some growth stimulating, immunomodulating and immunoprotective factors.

[The cultivation features, growth characteristics and oncogenicity of "serum-free" populations of transformed fibroblasts]. / Osobennosti kul'tivirovaniia, kharakteristiki rosta i onkogennost' "bessyvorotochnykh" populiatsii transformirovannykh fibroblastov.

The authors report about five previously selected cell lines of transformed mouse and rat fibroblasts, capable of growing in the medium lacking exogenous proteins. In contrast to cells of the parental lines, the cells under investigation have the ability to semi-suspension growth pattern and demonstrate an increasing sensitivity to damaging action of trypsin. The lowering of the autological conditioned medium (CM) concentration below 10-15%, as well as the reducing of the initial population density to 0.2 x 10(4)-0.4 x 10(5) cells per 1 cm2, resulted in inhibition of the serum-free cell proliferation. The feeder cultures of the serum-free cells immobilized by X-ray are able to stimulate proliferation of the intact autological cells in medium with semi-liquid agar in the presence of serum. CMs of the serum-free cell cultures stimulated the DNA synthesis in stationary cultures of NIH 3T3 cells in the absence of serum. The loss of dependence on serum was accompanied by a considerable broadening of the metionine-containing protein spectrum in the culture medium of the transformed rat embryo fibroblasts. In addition, it was demonstrated that selection for independence on exogenous proteins was accompanied by the loss of oncogenicity in both sensitive and resistant to cytotoxic effect of ethidium bromide serum-free cell lines, derived from L929 cell line. On the contrary, the serum-free variants from spontaneously transformed rat embryo fibroblasts as well as from C3H 10T1/2 cell line exhibit an enhanced oncogenicity as compared with the parental cells.(ABSTRACT TRUNCATED AT 250 WORDS)

[The isolation and characteristics of monoclonal antibodies to the HNK-1 antigen of natural killers]. / Poluchenie i kharakteristika monoklonal'nykh antitel k HNK-1-antigenu estestvennykh killerov.

Monoclonal antibodies to the HNK-1 differentiated antigen of natural killer cells have been obtained. A glycoprotein of the white human brain connected with myelin was used as an antigen for immunization of mice. The monoclonal antibodies obtained are shown to reduce the cytotoxic activity of a human blood mononuclear fraction as much as by 65% in relation to the human lymphoblastoma cell culture K-562. Their interaction with the surface antigen of mononuclears was shown by immunofluorescent method. Monoclonal antibodies belong to the class of immunoglobulins M.

[The autocrine regulation of proliferation in cell cultures. I. A study of the heparin-binding factors with growth-stimulating activity contained in media conditioned with transformed rat fibroblasts]. / Autokrinnaia reguliatsiia proliferatsii v kul'turakh kletok. I. Issledovanie geparinsviazyvaiushchikh faktorov s rostostimuliruiushchei aktivnost'iu, soderzhashchikhsia v sredakh, konditsionirovannykh transformirovannymi fibroblastami krysy.

A study was made of the medium conditioned by spontaneously transformed rat embryo fibroblasts of line Rec1-sf, which are capable of unlimited reproduction in medium free of serum and of other endogenous growth factors (c-medium). Addition of c-medium to stationary cultures of nontransformed rat embryo fibroblasts (REF), spontaneously transformed REF (line Rec1), and cells of Rec1-sf stimulated the incorporation of 3H-thymidine by 1.5-6 times. SDS-polyacrilamide-gel electrophoresis of proteins, marked by 35S-metionine of c-medium of the cell line Rec 1-sf, demonstrated that this medium had proteins with molecular mass from 10 to 110 kDa. The fractionating divisible by 100 ultra-concentrates of c-medium with utilization of heparin-sepharose allowed to isolate two types of heparin-binding proteins. The proteins of the first type took about 5% of all the proteins of c-medium; they were eluted with 1.1 M NaCl and stimulated the incorporation of 3H-thymidine in REF, Rec1 and Rec1-sf cultures by 1.3-1.9 times. The second type proteins took about 1% of all the proteins of c-medium and were eluted with 2M NaCl, and, like the main endogenic basic growth factor of fibroblasts, stimulated the incorporation of 3H-thymidine into REF and Rec1-sf, but not into the culture of Rec1 line cells. The results obtained are discussed in terms of a hypothesis of autocrine regulation of cell proliferation.

[The regulation of splenocyte natural killer activity in C3HA mice in the early period after hepatoma 22a transplantation]. / Reguliatsiia estestvennoi killernoi aktivnosti splenotsitov myshei linii C3HA v rannie sroki posle transplantatsii gepatomy 22a.

A decrease of the natural killer activity (NKA) of C3HA splenocytes after transplantation of hepatoma 22a cells was connected to some extent with the suppressor action of macrophages and T-lymphocytes. Elimination of the macrophages from the splenocyte suspension led to the increase of NKA. Addition of the macrophages to the nonadherent cells resulted in the NKA inhibition to the level of nonfractionated splenocytes. T-cell elimination with anti-T-serum and a complement led to the increase of NKA as compared to NKA of the nonfractionated splenocytes.

[Natural killer activity and sensitization of the lymphocytes to tissue antigens in lung cancer]. / Estestvennaia killernaia aktivnost' i sensibilizatsiia k tkanevym antigenam limfotsitov pri rake legkogo.

A decreased natural killer activity was observed in patients with lung cancer as the disease progressed. Surgical intervention results in its elevation at the IIId stage, while the application of cytostatics or irradiation are efficient at the IIId and IInd stages. Pronounced sensibilization of the peripheral blood lymphocytes in cancer patients to normal and cancer tissue antigens was demonstrated with the help of the leukocyte adhesion inhibition test.

[Natural killer activity of splenocytes of C3HA mice in the early period after transplantation of hepatoma 22a cells]. / Estestvennaia killernaia aktivnost' splenotsitov myshei linii C3HA v rannie sroki posle transplantatsii kletok gepatomy 22a.

It is shown that during 10 days after transplantation of syngenic 22a hepatoma cells to C3HA mice, wave-like fluctuations of the natural killer cell (NK) activity of splenocytes take place. Xenogenic (K-562) and allogenic (AC-1) cells are used as target cells in the 18th hour 3H-uridine cytotoxic test. A significant increase in the NK-activity on the 2nd-3rd and 7-9th days after tumour cells transplantation is observed. Elimination of adherent cells from suspension of splenocytes lead to an increase in the NK-activity especially on the 7th-9th days.

[Sensitization of murine splenocytes to normal tissue antigens and natural killer activity. II. The effect of immunization with allogeneic and xenogeneic tissues]. / Sensibilizatsiia splenotsitov myshei k normal'nym tkanevym antigenam i estestvennaia killernaia aktivnost'. II. Vliianie immunizatsii allogennymi i ksenogennymi tkaniami.

Immunization of C3HA mice with allogeneic tissue (liver of DBA mice) leads to wave-like variability in the natural killer activity of their recipient splenocytes. This variability is similar to that of NK-activity due to syngeneic immunization and is characterized by a sharp increase on days 1, 3-4 and 6 after immunization. Xenogeneic immunization exerts no influence on NK-activity, if compared with the injection of 0.14 M NaCl.

[Control of cell multiplication in vitro. V. The dependence of cell multiplication on the ganglioside content]. / Kontrol' razmnozheniia kletok in vitro. V. Zavisimost' razmnozheniia kletok ot soderzhaniia v nikh gangliozidov.

The concentration and composition of gangliosides in the cells of L-line and its clones--L-2 and Lebr 1/1, with values of saturation densities being 0.9, 1.6 and 3.2 X 10(5) cells/mc2, respectively, were compared. The transition of populations in the stationary growth phase was shown to be accompanied by an increment of ganglioside concentration and accumulation of di- and three-sialogangliosides. The data obtained are discussed in connection with the previously published results about changing intracellular cAMP content and conA-mediated agglutination at density-dependent growth inhibition L-cell variants.

[Cyclic adenosine monophosphate and the enzyme activity of its metabolism in L cells sensitive and resistant to the cytotoxic action of ethidium bromide]. / Tsiklicheskii adenozinmonofosfat i aktivnost' fermentov ego metabolizma v kletkakh linii L, chuvstvitel'nykh i ustoichivykh k tsitotoksicheskomu deistviiu bromistogo étidiia.

Using L-cells both sensitive and resistant to cytotoxic action of ethidium bromide (EB), a study was made of the intracellular level of cAMP, activities of adenylcyclase, phosphodiesterase and cAMP, liberated from cells into the surrounding medium. In EB resistant L-cells compared to EB sensitive ones, the higher level of cAMP with a decreased activity of adenylcyclase and an increased activity of the phosphodiesterase was shown to be associated with an impeded exit of cAMP from cells. It is suggested that the differences in cAMP levels in the EB sensitive and resistant cells are associated with the properties of cAMP-dependent protein kinases of these cells.

[Control of cell multiplication in vitro. II. Relationship between cell multiplication and cyclic AMP concentration]. / Kontrol' razmnozheniia kletok in vitro. II. Zavisimost' razmnozheniia kletok ot soderzhaniia v nikh tsiklicheskogo AMF.

Compairing non-transformed mouse fibroblasts and some L-cell variants, it was shown that the decrease of dependence on serum mitogenic factors, and the decease of sensitivity to the contact inhibition of growth do not always correlate with the decrease of intracellular cAMP content. The decrease of sensitivity of transformed cells to the factors limiting non-transformed cell growth in vivo might be also connected with a relative independence of the cell growth on the intracellular level of a cAMP.

[Control of cell proliferation in vitro. I. Relation of proliferation to blood serum factors]. / Kontrol' razmnozheniia kletok in vitro. I. Zavisimost' razmnozheniia ot faktorov syvorotki krovi.

The population of L-cells sensitive to the cytotoxic action of ethidium bromide (EB) has at least 2 cell types differing in some growth characteristics and in their dependence on serum factors, controlling saturation density of culture. L-cells, resistant to EB, differ from sensitive cells in many features studied and reveal the ability for a continuous and, probably, unlimited multiplication in serum-free medium.