Mitomycin-treated Cells Research Articles

Gene transfer of a secretable form of IL-15 in murine adenocarcinoma cells: Effects on tumorigenicity, metastatic potential and immune response

IL-15 is an immunostimulatory cytokine with IL-2-like activities. To exploit the potential role of IL-15 in cancer immuno-/gene therapy, we engineered murine TS/A cells with different IL-15 cDNA constructs. Significant IL-15 secretion was achieved only by the use of a modified cDNA encoding for an IL-15 pre-protein bearing the IgK light chain signal peptide. Different TS/A clones (TS/A IL-15 C6, C23, C29) producing 390 to 1,600 pg/ml biologically active IL-15 showed reduced tumorigenicity when implanted s.c. in syngeneic mice and significantly reduced metastatic potential by i.v. injection. Tumorigenicity of s.c. TS/A IL-15 was restored in animals depleted of CD8(+) lymphocytes or of natural killer cells and partially in CD4(+)-depleted mice. TS/A IL-15 cells displayed a significantly reduced growth rate by s.c. implant in nude mice. Also, >50% syngeneic animals rejecting TS/A IL-15 were resistant to a subsequent rechallenge with wild-type tumor (TS/Apc), indicating induction of protective immunity against TS/A tumor-associated antigens (TAAs). Cytolytic T lymphocyte (CTL) activity, specifically inhibited by anti-CD3 antibodies, was inducible in the splenocytes of TS/A IL-15-immunized animals by mixed lymphocyte/tumor culture (MLTC), and IFN-gamma was released in the supernatant of MLTC, mainly by CD8(+) cells. Immunohistochemistry of the TS/A IL-15 tumor area revealed the presence of an inflammatory infiltrate with predominant natural killer, macrophage, and granulocyte components and expression of IFN-gamma as a distinctive secondary cytokine. Use of TS/A IL-15 mitomycin-treated cells for therapeutic vaccination in experimental TS/A metastasis was effective in 60% of animals treated; these animals showed no metastatic tumor growth.

Mitogenic Effect of α1‐Microglobulin on Mouse Lymphocytes

Human alpha 1-m microglobulin (alpha 1-m), a low molecular weight plasma protein, was found to exert mitogenic effects on mouse lymphocytes from lymph nodes and spleen. The stimulatory effects appeared to be strain-restricted: alpha 1-m induced a varying degree of proliferation of lymphocytes from three strains, whereas one strain responded poorly. Experiments with lymphocyte subpopulations showed only weak stimulatory effects of alpha 1-m on purified T and B lymphocytes cultivated alone. The addition of mitomycin-treated cells of the other subpopulation could not restore the proliferative responses in either T or B lymphocytes. Strong stimulations were recorded only when both T and B lymphocytes were present, indicating that the T and B lymphocytes cooperate to achieve the proliferation. However, FACS studies on cultured splenocytes indicated that the proliferating cells are predominantly B lymphocytes. These data extend our earlier findings of a mitogenic effect of alpha 1-m on guinea pig lymphocytes. Furthermore, results were obtained indicating the presence of a receptor on mononuclear cells. Iodine-labelled alpha 1-m was bound to mononuclear cells prepared from spleens, and the binding could be blocked by an excess of non-labelled alpha 1-m. Scatchard plotting of the data gave an equilibrium constant of 0.7 x 10(5)/M for the binding between alpha 1-m and the receptor. Together with the documented inhibitory activity of alpha 1-m on antigen-driven proliferation of lymphocytes, these results suggest an immunoregulatory role for alpha 1-m.

Sensitivity of mitomycin-C treated McCoy cells for isolation ofChlamydia trachomatis from genital specimens

A new cell culture method was developed for the diagnostic isolation of Chlamydia trachomatis using McCoy cells pretreated with mitomycin-C. This drug acts on the cell by producing cross links between strands of DNA and results in the production of large, flat cells, which are similar to irradiated cells, and large chlamydial inclusions. In tests using a laboratory isolate of Chlamydia trachomatis, there were significantly more inclusions produced in mitomycin-treated monolayers than in cycloheximide-treated monolayers. Using clinical specimens, significantly more isolates were obtained in mitomycin-treated cells than in cycloheximide-treated cells. The new cell culture technique therefore offers a cell culture method which has the advantages of producing large inclusions similar to irradiated cells but with the convenience of drug treatment.

Presence of mitomycin resistant T cells in peripheral blood of normal individuals

T cell colonies can be easily grown from peripheral blood, and are an index of cellular immunocompetence. Mitomycin-treated T cells are used as stimulator cells in mixed lymphocyte reactions and as feeder cells for growth of B cell colonies, the assumption being that mitomycin prevents proliferation of T cells. We tested this assumption by comparing the proliferation of mitomycin-treated T cells in response to stimulation with phytohemagglutinin (PHA) with that of untreated T cells in liquid cultures and in T cell colony assay. We found that incorporation of tritiated thymidine by cells from 11 healthy individuals pretreated with 25, 50 and 100 μg/ml mitomycin C was reduced to 13, 11 and 8%, respectively, of that of untreated cells when stimulated by an optimal concentration of PHA (10 μg/ml) in liquid cultures. However, parallel experiments with aliquots of the same cells showed that pretreatment with 25, 50 or 100 μg/ml mitomycin C merely reduced T cell colonies to 49, 45 and 45%, respectively, of untreated cells. In five additional experiments mitomycin 200 and 400 μg/ml reduced T cell colony numbers to 47 and 60%, respectively. Treatment of T cells with 9000 rad completely abolished T cell colony formation. Lower doses of radiation up to 6000 R did not abolish T cell colony formation, although it effectively blocked T cell proliferation to PHA in liquid cultures. 24 h preincubation of T cells with suboptimal doses of PHA and then treatment with mitomycin or radiation did not abolish T cell colony formation. T cells were recovered from the mitomycin-resistant T cell colonies and stimulated in liquid cultures with PHA, untreated or after exposure to 25 μg/ml mitomycin C. Incorporation of tritiated thymidine by the mitomycin-treated cells was reduced to 8% of the untreated controls. Our observations suggests: (1) There may be inaccuracies in B cell colony assays using mitomycin-treated T cells because of significant T cell colony formation. (2) There is a population of T cells in the peripheral blood of normal individuals which form colonies and are resistant to mitomycin.

A genetic approach to the study of mitomycin-induced lysis of Escherichia coli K-12 strains which produce colicin E2.

A 2,250 base pair Sau3A restriction digest fragment of the colicin plasmid ColE2-P9 was shown to contain all the information necessary for colicin production, immunity to colicin E2 and mitomycin-induced lysis in host cells. Transposon Tn5 insertions in the colicin structural gene (ceaB) completely abolished colicin production and mitomycin-induced immunity protein production and lysis. This indicates the existence of a SOS-regulated colicin E2 operon in which ceaB:: Tn5 mutations exert a polar effect on genes concerened with lysis and production of immunity protein. However, cells harbouring these plasmids were immune to colicin E2, a phenomenon which is probably explained by the existence of an internal, SOS-independent promotor in front of the immunity gene (ceiB). A Tn5 insertion in a site (celB) removed from ceaB and ceiB produced a colicin+ immunity+ lysis- phenotype in host cells treated with mitomycin. These cells produced abnormally low amounts of colicin both in the presence and absence of mitomycin induction. Another difference between these cells and those harbouring wild-type ColE2 plasmids is that the vast majority of the colicin they produced remained within the cells. Cells carrying the wild-type ColE2 plasmid were shown to produce four additional polypeptides when grown in the presence of mitomycin. The two small polypeptides (Mr 5,000 and 3,000) were specifically absent from mitomycin-treated cells harbouring the plasmid with the celB:: Tn5 mutation, whereas all four polypeptides (the other two being the colicin and immunity proteins) were not detected in mitomycin-treated cultures of cells harbouring the plasmids with ceaB:: Tn5 mutations. The possible role of the Mr 5,0000 and 3,000 polypeptides in the regulation of colicin synthesis, cell lysis and colicin release is discussed.

ANTIGEN-SPECIFIC SUPPRESSOR CELLS IN SUBCLINICAL LEPROSY INFECTION

A two-stage in-vitro culture system was used to assay cells which suppress the lymphoproliferative response to Mycobacterium leprae (ML). Responses to ML, purified protein derivative of tuberculin, and streptokinase-streptodornase were preferentially suppressed by mitomycin-treated cells which had been primed with the same antigen in a 7-day primary culture. Healthy subjects exposed to leprosy for more than 3 years showed strong suppression of the response to ML antigens (11 of 12 showed more than 40% suppression), whereas those exposed for 3 months to 3 years showed much less suppression (12 of 15 showed less than 40% suppression). The in-vitro generation of strong ML-specific suppression may reflect the maturation of a well-regulated and protective immune response. However, premature induction and in-vivo activation of these suppressor cells could predispose to disseminated (lepromatous) forms of leprosy. With this assay it would be possible to assess the ability of proposed leprosy vaccines to engage strongly the regulatory network controlling the immune response to ML in the same way as long-term exposure to the natural infection.

Significance of cytotoxic lymphocytes after various immunizing procedures in a virus-induced non-producer syngeneic system: correlation between in vitro and in vivo lytic activity.

An originally virus-induced, non-producer tumour system has been studied in relation to humoral and cellular cytotoxic responses to transplantation and other immunization techniques. In all experimental groups cytotoxic lymphocytes (CTL) were observed either directly or after mixed culture of lymphocytes and tumour cells (MLTC). Except for C'-dependent cytotoxic antibodies in mice immunized by irradiated cells, no antibody-mediated cytotoxicity was observed. In 2 protocols (transplantation and immunization by mitomycin-treated cells) CTL in vitro were not protective. In a third protocol (immunization by irradiated cells) CTL afforded partial protection and other factors appeared to be involved. The best in vivo protection was induced by immunization consequent on early surgical removal of a small number of transplanted tumour cells. This study provides lines of evidence for the effectiveness of protection supplied by CTL in well-defined conditions. Comparison with other modes of immunization indicated that these conditions were related to the quantity and to the characteristics of antigen involved.

Further Characterization of the Circulating Cell in Chronic Lymphocytic Leukemia

Peripheral lymphocytes from normal individuals and from patients with chronic lymphocytic leukemia (CLL) were cultured in vitro for 1-7 days. The growth response to phytohemagglutinin (PHA) was quantitated by the incorporation of tritiated uridine into RNA nucleotide during a 2-hr pulse with the radioisotope. While the maximum response in PHA-stimulated normal cultures appeared at 2-3 days, CLL cultures required 5-7 days to develop their maximal response, which was 50%-60% of the normal magnitude. Dilution of the number of normally reactive lymphocytes by culturing them with totally unreactive, mitomycin-treated cells produced a normal 72-hr maximal response, no matter what proportion of unreactive cells was included in the PHA-stimulated cultures. In addition, the response of peripheral lymphocytes from patients with myeloblastic leukemia, where large numbers of unreactive myeloblasts diluted the normal small lymphocytes, a depressed reaction occurred at the anticipated 2-3 days. Nylon fiber-adherent lymphocytes consisting of 85% immunoglobulin (Ig)-bearing cells responded minimally to PHA, but showed no evidence of a delay. When isolated from CLL patients, both fiber-adherent cells (Ig-bearing) as well as non-fiber-adherent (sheep erythrocyterosetting) cells responded to PHA in a delayed fashion. Similarly, a case of CLL, in which 93.5% of the circulating lymphocytes bore sheep red blood cell receptors, showed its peak response to PHA at 7 days. Therefore, using surface marker criteria considered characteristic of normal T cells and B cells, the delayed response to PHA on the part of CLL lymphocytes was independent of thymic or nonthymic origin.

Proposed mechanism of bacteriophage lambda induction: acquisition of binding sites for lambda repressor by DNA of the host.

Interference with the in vitro binding of lambda phage repressor to lambda operator DNA was observed when Escherichia coli DNA containing the following lesions was present in the reaction mixture: (a) DNA with single-strand breaks from pancreatic DNase (nicked DNA); (B) DNA isolated from thymine-straved cells; (c) DNA from ultraviolet-treated cells; (d) DNA of mitomycin-treated cells; and (e) DNA from a temperature-sensitive ligase mutant after 1 hr at 42 degrees. Normal E. coli DNA did not interfere. Binding of lambda cIing-minus repressor to operator DNA was not affected by E. coli DNA with lesions. DNAs from cells treated with increasing doses of mitomycin were proportionately more effective in competition for repressor, suggesting increasing binding sites per unit of DNA. A general model of virus induction is proposed, based on binding affinity of ultraviolet-sensitive repressors for single-strand breaks in the host DNA. The model is extended also to the presumptive repressor of cell division.

Comparison of Irradiated and Mitomycin-Treated Mouse Spleen Cells As Stimulating Cells in Mixed Lymphocyte Cultures and in vitro Sensitization

Abstract Mitomycin-treated and irradiated mouse spleen cells were compared as stimulating cells in allogeneic mixed lymphocyte culture (MLC) and as sensitizing cells in in vitro allogeneic sensitization. C57BL/6 and BALB/C spleen cells were irradiated at doses up to 5000 rads or treated with mitomycin C to inhibit DNA synthesis. Irradiated cells, as well as mitomycin-treated cells, were efficient both in stimulating MLC and in sensitizing in vitro.

Production of blastogenic factor by mitomycin-treated cells in mixed lymphocyte cultures.

Production of blastogenic factor by mitomycin-treated cells in mixed lymphocyte cultures.

Enhancement of the mixed lymphocyte response in the mouse by addition of thymocytes

Synthesis of DNA by mixtures of mouse lymph node and thymic cells was studied in vitro using mitomycin-treated allogeneic spleen cells as stimulator cells. The tests were performed to see whether there occurs a similar cell synergy during this reaction as has been reported during the in vivo graft-vs-host response. It was observed that mixtures of thymocytes and lymph node cells give higher incorporations of isotope-labelled thymidine than can be explained by a pure additive effect of the two cell populations tested separately. This enhancement of the reactivity was more pronounced using combinations of lymph node cells and medullary thymocytes obtained from cortisone-treated donors. Enhancement was also noted between lymph node cells and spleen cells. Blocking of the capacity of lymph node cells to synthesize DNA by treatment with mitomycin abolished this enhanced activity when mixed with thymic cells. On the contrary, mitomycin treatment of thymocytes did not abolish their capacity to increase the reactivity when mixed with normal lymph node cells. Thymocytes, which were unresponsive to the mitomycin-treated cells for genetic reasons, were also found to increase DNA synthesis when combined with lymph node cells. The mechanism by which thymocytes increase DNA synthesis of lymph node cells is not clear, but the results show that they have to be present during the reaction, since culture medium “conditioned” by thymocytes did not exhibit any enhanced capacity to promote a mixed lymphocyte reaction of lymph node cells. The results are thus in agreement with the findings obtained by others showing that mixtures of lymph node cells and thymic cells yield higher immunological reactivities in vivo against foreign transplantation, antigens than can be explained by a pure additive effect of the reactivities by the two cell populations tested separately. However, in contrast to these findings, the thymic cells do not have to be able to synthesize DNA or to react against the foreign cells in vitro to yield an enhanced response when mixed with lymph node cells.

Histoincompatibility Recognition Reaction by Irradiated or Mitomycin-Treated Parental Cells against Hybrid Spleen Cells Produces Hybrid Cell Proliferation and Illusion of Reaction of Hybrid against Parent

Abstract The addition of heavily irradiated or mitomycin-treated spleen cells to cultures containing syngeneic spleen cells and foreign erythrocytes may stimulate to some extent the hemolytic plaque-forming cell responses of these cultures, but the addition of semi-allogeneic irradiated or mitomycin-treated spleen cells may cause much greater stimulation. This semi-allogeneic stimulation is completely reciprocal; the plaque-forming cell responses of F1 hybrid spleen cell cultures often are stimulated as much after the addition of non-proliferating parental cells as are the responses of parental spleen cell cultures after the addition of non-proliferating F1 hybrid cells. The possibility that an in vitro manifestation of hybrid resistance might be involved was ruled out. It was found that irradiated or mitomycin-treated cells, although unable to proliferate, were still able to initiate a recognition reaction against histoincompatible cells, releasing in the process a soluble factor which stimulated proliferation of cells of the antibody-forming series. The significance of this finding for “one-way” mixed lymphocyte reactions is discussed.

Lymphocyte transformation in vitro: V. Thymidine incorporation into unstimulated lymphocytes

In vitro conditions and kinetics of 14C-thymidine incorporation into unstimulated lymphocytes were studied. Lymphocytes cultured during 3 to 9 days displayed a progressive increase of thymidine uptake with time. The addition of varying numbers of autologous mitomycin-treated lymphocytes to cultures containing a fixed number of untreated lymphocytes markedly enhanced thymidine uptake per non-mitomycin-treated lymphocytes. In the latter cultures a sharp rise of thymidine uptake between the 7th day of culture was seen. Supernatants of non-stimulated lymphocytes, whether mitomycin-treated or not, showed no stimulating effect on autologous normal cells in these experiments. Although the mechanism of the enhancing effect of mitomycin-treated autologous lymphocytes remains unclear, it obviously may interfere in culture experiments in which the antigen specific stimulatory effect of mitomycin-treated cells is measured, which can only be detected by including the appropriate controls.

In vitro stimulation of mouse thymus cells by PHA and allogeneic cells

The in vitro reactivity of mouse thymus cells to phytohaemagglutinin (PHA) and allogeneic cells has been investigated using the incorporation of tritiated thymidine as a marker for cellular stimulation. It was observed that thymus cells from untreated donors were stimulated to a very slight extent in the mixed leucocyte culture (MLC) and by PHA compared to cells from lymph nodes and spleen. However, highly reactive thymic cells could be obtained by treating the donor animals with cortisone acetate. This treatment also enhanced the reactivity of the animals' lymph node cells, but the increase was small relative to thymus cells. Cortisone-resistant thymic cells cultured together with allogeneic mitomycin-treated cells were found to be transformed into killer cells. These cells could specifically kill fibroblasts in vitro carrying the same major histocompatibility antigens as the mitomycin-treated cells. The results strongly suggest that the cells in the thymus which can be stimulated by PHA or allogeneic cells are part of the relatively cortisone-resistant fraction of the thymus cell population.

Cellular Recognition by Mouse Lymphocytes in Vitro

Abstract The in vitro stimulation of mouse spleen cells by foreign histocompatibility antigens is described. Spleen cells from mice less than 3 weeks of age could not be stimulated in mixed lymphocyte cultures. They were also deficient in their ability to stimulate adult allogeneic cells in mixed lymphocyte cultures. The ability of the cells to be stimulated by histocompatibility antigens was correlated in time with their ability to stimulate allogeneic cells. Stimulation of mouse spleen cells was accomplished by allogeneic spleen cells with differences at the H-2 locus or at the H-3, H-13 loci, and with soluble H-2 antigen. F1 hybrid spleen cells were stimulated by parental strain spleen cells or by parental strain red blood cell stroma. Immunization with allogeneic spleen cells was found to transiently affect the degree of stimulation obtained in mixed lymphocyte cultures of spleen cells from the immunized mice mixed with mitomycin-treated cells of the strain used for immunization. The cells from immunized animals also showed an increased degree of stimulation when reacted in MLC with allogeneic cells sharing some of the H-2 antigens of the strain used for the immunization. Immunization did not affect the degree of stimulation obtained in MLC of cells from strains with weak histocompatibility differences. The possible explanations of in vitro lymphocyte stimulation by histocompatibility antigens of allogeneic or semi-allogeneic cells are discussed.

Thymidine incorporation in mixed cultures of spleen cells from mice of differing H-2 types.

SUMMARY Normal murine spleen cells were cultured with syngeneic, allogeneic, or hemiallogeneic spleen cells that had been treated with mitomycin- C; subsequent incorporation of tritiated thymidine was measured. Cell donors were genetically alike except for differences at the H-2 locus. When mitomycin-treated cells were mixed with normal cells, thymidine incorporation in allogeneic cultures significantly exceeded that in hemiallogeneic cultures; incorporation in both allogeneic and hemiallogeneic cultures was significantly greater than in syngeneic cultures. No significant difference could be demonstrated between groups of parent-vs.-Fi and Fr-vs.-parent cultures, nor between reciprocal allogeneic combinations. The results support the conclusion that antigenic disparity can induce DNA synthesis in splenic cells in mixed cultures. Conventional immunological incompatibility alone is insufficient to explain all of the results. The ability of the mixed-culture test to distinguish homozygous and hemizygous differences in dosage of antigens did not extend to the recognition of differences in number or in strength of antigens defined by serological tests.

Post-treatment breakage of mitomycin C induced cross-links in deoxyribonucleic acid of Escherichia coli

The renaturation following rapid cooling of heat-denatured DNA extracted from mitomycin-treated Escherichia coli is demonstrated. When mitomycin-treated cells of E. coli strain B are incubated, following treatment in a growing medium, but not in a non-growing medium, it is no longer renaturable after heat denaturation. This is interpreted to mean that the interstrand cross-links in DNA caused by mitomycin are broken during post-treatment incubation. The cross-linked DNA of mitomycin-treated mutants of E. coli, lacking host-cell or dark reactivating enzymes is not modified by post-treatment incubation. Sensitivity to mitomycin is to some extent, but not entirely, correlated with the absence of host-cell reactivating enzymes and their effect on cross-linked DNA.