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

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.     

Switch in the synthesis from IgM to IgG in the human lymphoblastoid cell line RPMI-6410t as affected by human sera

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.     

Hybridomas synthesizing monoclonal antibodies to surface antigens of human lymphocytes

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.     

Growth factor production and autocrine mechanism of cell proliferation regulation in the RPMI-6410t lymphoblastoid line

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.     

Growth-stimulating, cytostatic and cytotoxic activity of the secretory products of the RPMI-6410t human B-cell lymphoblastoid line

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).     

The biological action spectrum of the secretory products of the human B-cell lymphoblastoid line 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.     

人肝癌细胞系HC-108的建立及其生物学特征

HC108细胞系为贴壁生长具有上皮细胞形态特征的分化程度较高的人肝癌细胞。AFP分泌阴性,细胞平均倍增时间为32.21小时,细胞分裂指数为27.75‰,软琼脂克隆形成率为5.0％,能形成裸鼠移植瘤,支原体为阴性。染色体和流式细胞术分析为超二倍体核型,17号染色体改变最为明显。     

Autogenous production of growth factor as a mechanism of the cellular multiplication of the lymphoblastoid line 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.     

Establishment and characterization of a CA19-9 producing human gastric cancer cell line, STKM-1

A human gastric cancer cell line, STKM-1, was established from the malignant cells in pleural effusion of a 41-year-old female patient. The primary gastric cancer revealed histologically a poorly differentiated adenocarcinoma. The cells have been cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum and grew as monolayers following a doubling time of 31.4 hour at passage 30. The mode of chromosome number was 52. The STKM-1 cell was tumorigenic in nude mice. The STKM-1 cell cultured in vitro secreted CA19-9, into the medium as a tumor marker. Cells in tumors grown in nude mice were immunohistochemically recognized positively by anti-CA19-9 antibody. The STKM-1 will provide a useful information to clarify the mechanism of CA19-9 secretion.     

Clones of the lymphoblastoid line RPMI-6410t requiring an exogenous growth factor

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.     

Analysis of replication patterns in chromosomes of normal Chinese hamster and its cell lines

Replication patterns of the normal male Chinese hamster chromosomes and the three cell lines CHW, 1102 and 1103, were determined using fluorescent, plus Giemsa or acridine orange, techniques. The individual chromosomes or chromosomal segments were consistent in the replication patterns of normal Chinese hamster chromosomes and all the transformed cell lines. Late DNA replication was regularly identified in the long arm of the X chromosome, the entire Y chromosome, the short arms of chromosomes 6 and 7, and the paracentromeric regions of chromosomes 8, 9 and 10. A similar consistency was demonstrated in the large late replicating areas of chromosomes X and Y. Each cell line had specific marker chromosomes by which the cell line was identified and their replication patterns have been described. The chromosome analysis in cell line 1103 indicated that chromosomes 2, 3, 8 and 9 were more stable than others, of which chromosome 2 was extremely stable. The markers M4 and M5 in cell line 1103 are very interesting. The cytogenetic behaviour of marker M4 indicated a new phenomenon of translocation by simple association. The marker chromosome M5 indicated that inactivation spread to the early replicating distal region. These cell lines are very useful tools for studying replication patterns and providing a basic understanding of mammalian cytogenetics.     

Galactocerebrosidase activity in somatic cell hybrids derived from twitcher mouse/control human fibroblasts is associated with human chromosome 17.

Somatic cell hybrids derived from twitcher mouse cells and from control human fibroblasts were selected by two different methods. One method utilized 6-thioguanine-resistant twitcher cells as a parental line and the other used neomycin-resistant control human fibroblasts as a parental line so that hybrid lines could be selected in either HAT or in G-418 medium, respectively. The hybrid lines were analyzed for galactocerebrosidase activity. Since the twitcher cell lines are deficient in galactocerebrosidase activity, the presence of this activity in these hybrid lines depends upon the presence of human chromosome contents. Both galactocerebrosidase-positive and -deficient hybrid lines were analyzed for their human chromosome contents by the use of isozyme markers. In hybrids derived from both selection methods the expression of galactocerebrosidase activity was associated with the presence of human chromosome 17 marker isozymes. This was confirmed cytogenetically by means of trypsin-banded Giemsa staining of intact human chromosome 17 in three galactocerebrosidase-positive hybrid lines.     

Clonal sublines of rat neurotumor RT4 and cell differentiation. VI. Chromosome analysis

The RT4 neurotumor cell system consists of clonally derived cell lines where a stem cell type segregates in vitro into three biochemically and morphologically different cell types, one glial and two neuronal types. This process has been termed cell-type conversion (M. Imada and N. Sueoka, 1978, Dev. Biol. 66, 97-108). Detailed cytogenetic analysis of the RT4 cell lines are described. Giemsa-banding analysis of 12 independent clonal isolates of the four different RT4 cell types showed a relatively stable karyotype. The stem cell line, RT4-AC, is diploid and most stable, and it has one 4q+ marker chromosome in place of a normal No. 4. This 4q+ marker was identified in all cell types of the RT4 system and was not observed in other cell lines of BDIX origin. The 4q+, therefore, is a chromosomal marker of the RT4 system. Consistent chromosome rearrangement was not found in any one of the cell-type conversions of the RT4-AC cells into the three derivative cell types. The relative stability of the karyotype of the different clonal isolates gives the RT4 system an advantage in studies of genetic regulation and expression of cell-type conversion in vitro. Also the 4q+ marker can be used to identify RT4 cells in coculture experiments or to distinguish RT4 cells in cases of suspected cell-line contamination.     

Effects of Citrus aurantifolia concentrated extract on the spontaneous proliferation of MDA-MB-453 and RPMI-8866 tumor cell lines.

The in vitro effects of concentrated lime juice (CLJ) extract on the spontaneous proliferation of a human breast carcinoma cell line (MDA-MB-453) and a human lymphoblastoid B cell line (RPMI-8866) were investigated. CLJ extract was prepared by freeze-drying fresh fruit juice and dialyzing the concentrated extract against phosphate buffered saline in order to deplete low molecular weight micronutrients such as flavonoids as well as adjusting the pH of the extract to the physiological range. The effects of different concentrations of the CLJ extract on the spontaneous proliferative responses of the cell lines were determined by 3H-thymidine incorporation after 24 hrs of incubation. CLJ extract had no significant effect on MDA-MB-453 cell line, however, using the concentrations of 125, 250, and 500 microg/ml of CLJ extract a significant inhibition of the spontaneous proliferation of RPMI-8866 cell line was detected (P < 0.05). Due to the protein nature of the biologically active macromolecules of the CLJ extract (Gharagozloo and Ghaderi, 2001), it is reasonable to assume that the protein components of the CLJ extract may have anti-proliferative effects on tumor cell lines.     

A cell hybrid and recombinant DNA library that facilitate identification of polymorphic loci in the vicinity of the Huntington disease gene.

Somatic cell hybrids were selected that retain a derivative chromosome 5 from an individual in which the p15.1-pter segment of chromosome 5 is replaced with the p15.1-pter segment of chromosome 4. Hybrids that retain this derivative chromosome exclusively were found to be positive for G8, a DNA marker closely linked to the Huntington disease gene on chromosome 4p. From one such hybrid, a segregant was isolated that had deleted the entire q arm of the derivative chromosome but retained the p arm intact as its only detectable human DNA. A complete recombinant DNA library was prepared from this cell line, and the inserts in approximately 1/3 of the recombinant phage with human DNA were shown to be derived from 4pter-4p15.1, which represents only approximately 1% of the total human genome. The cell hybrid and DNA library represent a rapid and efficient means to identify and isolate many polymorphic DNA markers close to and flanking the Huntington disease gene.     

Evidence for linkage between HLA antigens and receptors for complement components C3b and C3d in human-mouse hybrids

A linkage between HLA-A and HLA-B antigens and receptors for complement components C3b and C3d has been demonstrated by means of human-mouse somatic cell hybridization. In 26 hybrid clones formed between the human lymphoblastoid cell lines RPMI 6410 and Raji and the murine fibroblast line 1T22, HLA-A and HLA-B antigens and receptors for C3b and C3d segregated conjointly. No segregation of the C3b and C3d receptors from each other or from HLA antigens was observed. The results suggest that the genes for the C3b and C3d receptors are located on the same chromosome that encodes the major histocompatibility complex of the human.     

The effect of anoxia on anthracycline-induced DNA damage in the RPMI-6410 human lymphoblastoid cell line

The alkaline elution procedure developed by Kohn and co-workers was used with the RPMI-6410 cultured human lymphoblastoid cell line to examine the hypothesis that anthracycline-induced DNA strand scission is mediated by oxygen- or superoxide-derived free radicals. Hypoxia was induced by gassing with nitrogen containing 5% carbon dioxide and less than 4 ppm oxygen. Alkaline elution studies showed hypoxia was induced, as the oxygen enhancement ratios for DNA strand breaks was 2.4 and 2.6 for the 250 R +/- oxygen and the 500 R +/- oxygen (1 R = 2.58 x 10(-4) C/kg) experiments, respectively. The pattern of adriamycin-induced DNA strand breaks and cross-linking was not affected by hypoxia with 1-h adriamycin exposures between 0.05 and 1.0 microgram/ml. Similarly, 1-h exposures of N-trifluoroacetyladriamycin-14-valerate at 3 or 10 micrograms/mL gave essentially identical alkaline elution profiles in the presence or absence of oxygen. These results do not support the hypothesis that oxygen-derived radicals play a primary role in anthracycline-induced DNA strand breakage.     

Nucleolar organizing regions of normal Chinese hamster and CHW cell line chromosomes

The chromosome preparations from fibroblasts of normal male and female Chinese hamsters and the cell line CHW were stained with AgNO3. The silver stain was usually localized at the telomeres of autosomes. The marker chromosome M1 in the CHW cell line has Ag-NOR near the centre of the long arm, which indicates that either the long arms of two number 5 chromosomes fused at the telomeres or the intact telomeric region of one chromosome fused with one with a deleted telomere. The variation of Ag-NORs' number per cell and Ag-heteromorphism in chromosome number 4 were observed. The Ag-NORs of chromosome number 4 and 5 are in approximately the same position as the positive C-bands and these may play a role in the preservation of heterochromatin.     

Human alpha-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells.

We have used the genetic marker, adenine phosphoribosyl transferase (APRT), an enzyme known to be on human chromosome 16, to establish a method for the transfer of human α-globin genes into mouse erythroleukemia cells. Mouse erythroleukemia cells devoid of detectable levels of APRT were fused with fractions of human marrow enriched in human erythroid cells. The hybrid cells arising from this fusion were isolated in medium supplemented with aminopterin and thymidine, and used adenine as the sole purine source. This population of hybrid cells was dominated by cells (80%) in which human chromosome 16 was present. Human chromosomes 4, 5 and 6 were also found in these cells. The hybrid cells were then placed in medium supplemented with diaminopurine (DAP), which is lethal for cells containing APRT. Greater than 95% of the DAP-selected hybrid cells lacked human chromosome 16. Cytoplasmic RNA was extracted from the two hybrid cell populations and assayed by molecular hybridization for sequences coding for human α-globin. Carboxymethyl cellulose chromatography was used to study the level of synthesis of human a-globin in the hybrids. The original hybrid cell, which contained a high frequency of human chromosome 16, also contained high levels of human a-globin mRNA and human α-globin chains. Hybrid cells counter-selected in DAP and thus lacking human chromosome 16 were devoid of detectable levels of human APRT, human α-globin mRNA and human α-globin chains. This work shows that transfer of human chromosome 16 into the MEL cell is possible using a chromosomedependent, APRT-mediated method of gene transfer. Using this system in which expression of the human α-globin gene occurs, we were also able to confirm our earlier assignment of the human α-globin gene to human chromosome 16. This system may be of further use in identifying genetic elements governing expression of the human α-globin gene which can be carried with human chromosome 16 as it is donated to the mouse erythroleukemia cell by donor cells of different epigenotypes.     

Chromosomal changes in cell lines from mouse tumors induced by nickel sulfide and methylcholanthrene

Rhabdomyosarcomas were induced in mice by intramuscular injections of crystalline nickel sulfide and 3-methylcholanthrene. At early passage, karyotypes were performed by G-banding for four nickel sulfide cell lines and for three 3-methylcholanthrene cell lines. Six cell lines were near-diploid and one nickel sulfide line was near-tetraploid. Three of the nickel sulfide cell lines were characterized by a rearranged marker chromosome which was present in a majority of the cells of each line. The rearrangements leading to the formation of marker chromosomes were different in each nickel sulfide cell line but involved chromosome 4 in two of the nickel sulfide cell lines. Extra copies of chromosome 15 were present in two nickel sulfide cell lines. Possible rearrangement and/or gene activation was examined for the c-mos oncogene on chromosome 4 and the c-myc oncogene on chromosome 15, but no alteration or activation was observed. None of the 3-methylcholanthrene cell lines contained rearranged marker chromosomes; however, one MCA cell line did contain large numbers of double minutes. In all cell lines, minichromosomes (small atypical acrocentric chromosomes) were observed that contained distinct centromeric regions but no other G-positive bands.Abbreviations DHFR dihydrofolate reductase - MCA 3-methylcholanthrene - NS nickel sulfide