An autocrine growth factor constitutively produced by a human lymphoblastoid B-cell line is serologically related to lymphotoxin (TNF-beta).

Conditioned medium of a human lymphoblastoid B-cell line RPMI-6410t contains a factor sufficient for maintainance and growth of these cells. At the same time RPMI-6410t cells secrete a soluble factor cytotoxic towards mouse L929 cells. Production of these activities by RPMI-6410t cell line and its subclones is significantly enhanced after activation with phorbol mirystate acetate (PMA). Both activities can be neutralized by antiserum raised against recombinant lymphotoxin (rTNF-beta) but not by antibodies against tumor necrosis factor (rTNF-alpha). Northern analysis showed the presence of lymphotoxin mRNA which is further induced after PMA treatment. These data suggest that both autocrine growth factor and cytotoxic activities correspond to the same molecule(s) probably identical to 25 kD lymphotoxin (TNF-beta).

[Reversible suppression and activation of immunoglobulin genes in differentiated IgG+ sublines of human RPMI-6410t B-cells]. / Obratimaia supressiia i aktivatsiia genov immunoglobulinov v differentsirovannykh IgG+-subliniiakh B-kletok cheloveka RPMI-6410t.

Differentiated human B-cells of the IgG+ sublines obtained as a result of switching from IgM to IgG synthesis in the 6410t line and its IgM+ lines gradually reduce the level of IgM secretion after the inductor removal. IgG synthesis can be partially or fully recovered by treating the IgG+ sublines with a polyclonal activator of B-lymphocytes, lipopolysaccharide W from Gram negative bacteria. In the conditions of certain regulatory effects, differentiated IgG+ cells are capable to pass reversibly in the state of functional rest and synthesis initiation.

[The biological action spectrum of the secretory products of the human B-cell lymphoblastoid line RPMI-641Ot]. / Spektr biologicheskogo deistviia sekretornykh produktov limfoblastoidnoi linii B-kletok cheloveka RPMI-641Ot.

Secretory products of the lymphoblastoid line of human B-cells RPMI-6410t have a wide spectrum of biological activity. These products exert a growth-stimulating effect on B-cells of the 6410t strain obtained from the peripheral blood of a patient with acute myeloblastic leukemia and cytotoxic and cytostatic effects, respectively, on B-cells of the Raji and P3HR-I.G5 lines obtained from patients with Burkitt's lymphoma. They also exert a cytotoxic effect on murine L-cells. Biological activity is relieved at 70 degrees and at low and high pH.

[Instability of immunoglobulin expression during differentiation induction in the human lymphoblastoid cell line RPMI-6410t]. / Nestabil'nost' ékspressii immunoglobulinov pri induktsii differentsirovki v linii limfoblastoidnykh kletok cheloveka RPMI-6410t.

The induction of immunoglobulin heavy chain classes switch from IgM to IgG was demonstrated in vitro in cells of RPMI-6410t line. The IgG+-sublines, formed as a result of the switch are characterized by instability of IgG synthesis. After removal of the inductor from the growth environment, IgG+ cells gradually reduce the level of secreted IgG. Such a transition to the functional rest state is likely to be connected with the convertible Ig-gene activity suppression in IgG+ cells, since after their activation by LPS IgG-secretion is partly or completely restored. The IgG+-sublines obtained may serve a convenient model for investigating the Ig-gene expression regulation in differentiated human B-cells.

[Switch in the synthesis from IgM to IgG in the human lymphoblastoid cell line RPMI-6410t as affected by human sera]. / Perekliuchenie sinteza s IgM na IgG v limfoblastoidnoi linii kletok cheloveka RPMI-6410t pod deistviem syvorotok cheloveka.

The switch from IgM to IgG in lymphoblastoid cell line RPMI-6410t was induced by human sera. The factor inducing the switch was found in the human placental serum and in the serum of peripheral blood of healthy donors. The switch investigated is induced both in the initial line 6410t and in some IgM+ sublines derived from it. With the help of the cloning method some IgG+ sublines were developed with different IgG-synthesis levels from 6410t line and its IgM+ sublines after inducing the switch in them. Earlier another type of the switch induction from IgM to IgA was observed in the same line and its IgM+-sublines by the factors contained in some batches of fetal calf serum (FCSG+). Thus, the homogeneous IgM+ cell population is shown to be able to pass in vitro though two different stages of differentiation inherent to B lymphocytes in vivo.

[2 physiological factors selectively inducing 2 different stages of differentiation in the human lymphoblastoid cell line RPMI-6410t]. / Dva fiziologicheskikh faktora, izbiratel'no indutsiruiushchikh dve raznye stadii differentsirovki v linii limfoblastoidnykh kletok cheloveka RPMI-6410t.

A factor was discovered in the human serum, placental serum from the umbilical cord of pregnant women and sera of peripheral blood from some healthy donors, which induces one of the late stages of differentiation: switching of IgM to IgG synthesis in the RPMI-6410t line of human lymphoblastoid B-cells. Earlier we observed the induction of another stage of differentiation in the same cell line: switching of IgM to IgA synthesis under the influence of a factor from the serum of cattle foetuses. We have shown that both the stages of differentiation are induced by the above factors not only in the initial cell line but also in a number of IgM+-sublines obtained by cloning with limiting dilutions. It was, thus, established that there are factors of class-specific switching and the B-cells of a human lymphoblastoid line are capable to proceed in vitro through different stages of differentiation inherent in the B-lymphocytes in vivo.

[Growth-stimulating, cytostatic and cytotoxic activity of the secretory products of the RPMI-6410t human B-cell lymphoblastoid line]. / Roststimuliruiushchaia, tsitostaticheskaia i tsitotoksicheskaia aktivnost' sekretornykh produktov limfoblastnoi linii B-kletok cheloveka RPMI-6410t.

The cells of human lymphoblastoid line RPMI-6410t were shown to synthesize constitutively a factor(s) with different types of biological activity. The factor(s) stimulated the growth of both B cells 6410t, obtained from the blood of a patient with acute myeloblastic leukemia, and the human embryonic diploid fibroblasts. With B cell lines Raji and P3HR-1.G5, obtained from the patients with Burkitt's lymphoma. The growth factor(s) displayed cytotoxic and cytostatic effects, respectively. Growth-stimulating and cytotoxic activating of the factor were destroyed by a 15 hour exposure to low or high pH. The activity was stable within pH values of 6-8. With regard to heat stability, the activity destroyed at 70 degrees C within 1 hour but remained stable at 56 degrees C during 1 hour. The above factor(s) displayed biological activities similar to those of the previously known tumor necrosis factor (TNF).

[Growth factor production and autocrine mechanism of cell proliferation regulation in the RPMI-6410t lymphoblastoid line]. / Produktsiia faktora rosta i autokrinnyi mekhanizm reguliatsii proliferatsii kletok limfoblastoidnoi linii RPMI-6410t.

The human lymphoblastoid B-cell line RPMI-6410t was found to synthesize and secrete into the growth medium a factor necessary to maintain the reproduction of these cells. In the condition of low plating density (concentration 1-1000 cells per ml) cell proliferation can be maintained only in the presence of a definite dose of medium conditioned by 6410t cell growth under high concentration. Using such a medium guaranteed almost 100% cloning efficiency of these cells by the method of limiting dilutions. The cloning of 6410t cells in the presence of feeder cells, such as mouse splenocytes and peritoneal cells, failed. The 6410t cells were shown to bind specifically the growth factor secreted by them, thus suggesting the presence of a growth factor acceptor on their surface. With the help of special selective method some clones were derived which did not secrete growth factor but were likely to have growth factor acceptors on their surface. A comparison of growth properties of clones GF- and GF+ supported the idea of autocrine control of proliferation as one of the mechanisms of malignant cell transformation.

[Clones of the lymphoblastoid line RPMI-6410t requiring an exogenous growth factor]. / Klony limfoblastoidnoi linii RPMI-6410t s potrebnost'iu v ékzogennom faktore rosta.

It was earlier established that the RPMI-6410t cells, obtained from a patient with acute myeloblastemia, synthesized a growth factor which maintains their proliferation and had a specific receptor for this factor on their surface. The use of a medium conditioned by the 6410t cells made it possible to define conditions in which practically 100% efficiency cloning of these cells is attained by the method of limiting dilutions. In the present work, this method of cloning was applied to obtain from the 6410t strain clones which are characterized by a requirement for an exogenous growth factor. These clones, like the 6410t cells, have on their surface specific receptors but, unlike the parental cells, do not synthesize the growth factor and do not form colonies in soft agar, i. e. lose one of the features of malignancy. These facts agree with the published data according to which the proliferation of normal cells is regulated by exogenous growth factors and confirm a suggestion put forward in our previous work that the endocrine regulation of cell growth is one of the mechanisms of malignancy.

[Hybridomas synthesizing monoclonal antibodies to surface antigens of human lymphocytes]. / Gibridomy, sinteziruiushchie monoklonal'nye antitela k poverkhnostnym antigenam limfotsitov cheloveka.

Three types of hybridomas were obtained by fusion of murine myeloma cells (NSI-1-Ag4-1) with splenocytes from mice immunized with human lymphoblastoid cells (RPMI-6410t line, acute myeloblastic leukemia). Hybridomas of the first type synthesize monoclonal antibodies Ma-1, which interact with 6410t-cells, but are not bound to the cells of human Burkitt lymphoma-Raji. Raji cells contain HLA-DRw5 and -DRw6 antigens on cell surface but there are no HLA-A2, -B7 and -B12 antigens (specific for 6410t). Thus, Ma-1 are probably derected against some of HLA antigens of loci A or B. Hybridomas of the second type synthesize Ma-2 antibodies which react with 6410t and Raji cells, but are not bound to peripheral blood lymphocytes (PBL). We suppose that Ma-2 antibodies to tumor specific antigens which have common antigen determinants both for Raji and RPMI-6410t cells. The third type of hybridomas synthesizes monoclonal antibodies Ma-3 reacting with all the three types of target cells: 6410t, Raji, and PBL. Ma-3 seems to be directed against human species-specific lymphocyte antigens which remained in 6410t and Raji cells.

[Autogenous production of growth factor as a mechanism of the cellular multiplication of the lymphoblastoid line RPMI-6410t]. / Autogennaia produktsiia faktora rosta kak mekhanizm razmnozheniia kletok limfoblastoidnoi linii RPMI-6410t.

The cells of the lymphoblastoid strain, RPMI-6410t (from the blood of a patient suffering an acute myeloblast leukemia), were shown to synthesize constitutively and secrete into the culture medium a growth factor that maintains the reproduction of these cells. The 6410t cells were shown to bind specifically this factor and react to it by proliferation in the conditions of rarefied inoculation. The utilization of a medium conditioned by the 6410t cells provided almost 100% cloning of these cells when using the method of limiting dilutions in 96-well microplates at a density of one cell per well. The cloning of the 6410t cells without the conditioned medium with feeder cells (mouse splenocytes and peritoneal cells) failed. It is suggested that as a result of the second stage of malignant transformation the immortalized cell still requires an exogenous growth factor, unlike was considered earlier, but acquires the ability of producing an endogenous growth factor and, hence, escapes the environmental control.

[Induction of the switch in the synthesis of immunoglobulin classes in the RPM 1-6410t lymphoblast cell line and its clones as affected by fetal calf serum factors]. / Induktsiia perekliucheniia sinteza klassov immunoglobulinov v linii limfoblastoidnykh kletok RPM 1-6410t i ee klonakh pod deistviem faktorov émbrional'noi teliach'ei syvorotki.

We have found that human lymphoblastoid cell line RPMI-6410t is a biochemical mutant for gene of thymidine kinase and has chromosome markers in the karyotype. Thus, this cell line can be used as a partner in somatic hybridization, in particular for producing hybridomas, synthesizing human monoclonal antibodies. We have discovered that line RPMI-6410t carries HLA-A2, -B7 and -B12 antigens of human histocompatibility complex on the cell surface. The cell membrane of this cell line contains immunoglobulins of M and D classes. RPMI-6410t cells secrete IgM molecules. It is demonstrated that induction of the switch of immunoglobulin heavy-chain classes by the factors of foetal calf serum takes place in the cells of RPMI-6410t line. Thus, the corresponding stage of B-lymphocytes differentiation in vivo is reproduced in 6410t line in vitro.

[Biochemically labelled human lymphoblastoid cell line. I. Its karyotypic, growth and physiological characteristics]. / Biokhimicheski markirovannaia limfoblastoidnaia liniia kletok cheloveka. I. Kariotipicheskaia, rostovye i fiziologicheskie kharakteristiki.

A study was made of the properties of human lymphoid cell line RPMI-6410 derived from peripheral blood of a patient with acute myeloblastic leukaemia. The lymphoblastoid cell line was found to be resistant to 5-brom-deoxyuridine and to have a low thymidine kinase activity. The modal chromosome number for RPMI-6410 is 46-47 XY. The karyotype includes marker chromosomes: two large submetacentrics --M1 and M2, and two small acrocentrics--M3 and M4. Ways of marker chromosome formation are discussed. The properties of RPMI-6410 line make it possible to use it for somatic cell hybridization, in particular, for obtaining hybridoma synthesizing human monoclonal antibodies.

[Characteristics of an interspecies somatic hybrid (Chinese hamster X mouse)]. / Osobennosti mezhvidovogo somaticheskogo gibrida (kitaiskii khomiachokXmysh').

An interspecific somatic hybrid between the biochemically marked cell lines of Chinese hamster (M-15-1) and mouse (B-82) was obtained. Morphology of the hybrid, its karyotype characteristics in the 9-th and 22-nd passages, as well as kinetics of growth were studied. Periods of doubling of the hybrid cells, the Chinese hamster M-15-1 cells and the mouse B-82 cells proved to be 26, 18 and 21 h, respectively. It was revealed, that elimination of chromosomes took place in the course of the hybrid cells reproduction at the expense of two-arm chromosomes of both parental lines.

[Phage T4 partial diploids obtained with the method of DNA interrupted injection. II. The mapping technique based on the physical size measurement of the diploid region]. / Merozigoty faga T4. II. Metod kartirovaniia, osnovannyi na opredelenii fizicheskogo razmera diploidnoi oblasti

Populations of the E. coli B mixedly infected with the whole chromosome of the amber mutant in respect to two genes under consideration and with a chromosome fragment of the phage T4 "wild" type are examined. The fragment length with which both of the amber mutants defect functions are compensated is determined to measure physical distance between the genes. Distances so obtained do not depend on recombination frequencies and are shown to be additive and "complementary". The latter property means that the sum of the smaller and "complementary", larger distances between two genes on the circular map equals the complete linkage group quantity. Moreover, a "complementary", larger distance is measured directly and independently on a smaller one, whereas current mapping technique fail to do so. The method enables to evaluate a size of rather long phage T4 gene region, for example gene 43, i. e. the structural DNA polymerase gene. Phage T4 genes 34 and 35 are shown to have a common start region for transcription and translation, i. e. they belong to the same operon. In consequences the circular map of physical distances between phage T4 genes is constructed.

[Phage T4 partial diploidy obtained with the method of DNA interrupted injection. I. Analysis of the genetic structure and phage progeny reproduction process]. / Merozigoty faga T4. I. Analiz geneticheskoi struktury i issledovanie protessa reproduktsii fagovogo potomstva

Phage T4 chromosome fragmentation is shown to take place when DNA injection is interrupted, a fragment length being strictly controlled by the interval from the moment of adsorbtion till the moment of an interruption. Populations of the bacteria cells infected by the phage T4 partial diploids are produced with the method of DNA interrupted injection. In the population a merodiploid involves some phage T4 amber mutant and a phage "wild" type chromosome fragment of the size controlled. To construct merodiploids the amber mutant in gene 43 and the mutant in gene 32 with the higher and the lower recombination frequency, accordingly, are used. Every merodiploid which is the heterozygote by one of these genes or which is the heterozygote by the late genes is determined to reproduce mixed phage progeny. Both the mean of the burst and the parent genotypes ratio in progeny either in the E. coli CR-63 cells or in the E. coli B depend on neither the heterozygote genetic structure nor the diploid region size. The results obtained conclude that phage genes express their function in the small fragments and the fragment recombination with the mutant partner whole chromosome follows their autonomous replication.

[Cyclic rearrangements in the incomplete genome of T4D phage transferred into a cell during a injections of DNA]. / Tsiklicheskie perestanovki v nepolnykh genomakh faga T4D, perenosimykh v kletku pri prebyvanii in"ektsii DNK

The genome structure of phage T4 chromosome fragments obtained by the method of DNA interrupted injection is studied directly. Anfragment population is shown by homogeneous in sizes. The fragment sizes are proved to the indentical to sizes. The fragment sizes are proved to be identical to sizes expected from the method conditions. The individual fragment genomes are shown to be cyclic gene permutations uniformly distributed on the map of physical distances between genes. The results suggest that any genetic marker has equal probabilities to be situated at the different distances from the individual particle of chromosome ends in a T4 D particles population.