Expression, tissue distribution, and cellular localization of the antiapoptotic TIP-B1 protein.

TIP-B1 is a novel 27-kDa protein isolated from the cytosol of tumor necrosis factor (TNF)-stimulated cells. Cells preincubated with TIP-B1 are protected from TNF-induced apoptosis. This study showed that, as with normal fibroblasts and U937 histiocytic lymphoma, human MCF7 mammary adenocarcinoma cells were protected from TNF in a concentration-dependent manner by pretreatment with either TNF or purified TIP-B1. Immunoblot and immunohistochemical analyses indicated expression of both TIP-B1 mRNA and protein in MCF7 cells and heart, kidney, brain, liver, ovary, uterus, thymus, spleen, lymph node, and mammary gland cells throughout their development. Expression of TIP-B1 was heterogeneous, with staining of specific cell types within tissues. Based on the ability of TIP-B1 to protect both normal and tumor cells from TNF-induced apoptosis and its broad tissue distribution, with expression only in select cells within those tissues, a role for TIP-B1 in the regulation of TNF-induced effects is strongly indicated.

Protective specific immunity induced by doxorubicin plus TNF-alpha combination treatment of EL4 lymphoma-bearing C57BL/6 mice.

The therapeutic efficacy of a single (day 8), moderate dose (4 mg/kg, i.v.) of doxorubicin (DOX, Adriamycin) combined with recombinant human TNF-alpha (3 different doses and 5 different schedules, i.v.) was evaluated in C57BL/6 mice bearing an implant (s.c.) of the DOX-sensitive, TNF-alpha-resistant EL4 lymphoma. In parallel to monitoring survival, the levels of several host anti-tumor cytolytic effector functions of splenocytes and thymocytes were evaluated throughout the treatment period and in long-term survivors (LTS). DOX treatment alone resulted in a moderate (approx. 20%) increase in life span but no cures. TNF-alpha alone, at any tested dose or schedule, had little or no positive effect on survival. The combinations of DOX and TNF-alpha were only slightly better than DOX alone with respect to the time to death of mice that died (approx. 29% increase); however, each of the combinations involving 1,000 U TNF-alpha/injection produced a fraction (20% to 80%) of LTS. The host defense activities examined included those of splenic and thymic cytolytic T lymphocytes (CTL) and lymphokine-activated killer cells as well as splenic tumoricidal macrophages. Although most activities were modulated by tumor growth and/or treatment, only CTL responsiveness appeared to correlate with survival. CTL activity in the treated groups with LTS was significantly higher than in control groups late in the treatment period. Finally, ex vivo analyses of splenocytes and thymocytes together with the rejection of implanted tumor at 17 months established that LTS displayed specific long-term immune memory.

Anticancer drugs plus cytokines: immunodulation based therapies of mouse tumors.

As demonstrated in this laboratory, several cytotoxic anticancer agents have immunomodulating effects at relatively low doses and, in combination with non-toxic doses of certain cytokines, can exert immunity-dependent curative effects in mouse tumor models. Thus, adriamycin (Dox) has been shown to enhance the activation of macrophages with associated increases of IL1 and TNF production, to stimulate the production of IL2 and the development and action of CTLs. In the EL4 lymphoma C57BL/6 mouse model, combinations of appropriate regimens of Dox plus IL2 or TNF induce cures and the long-term survivors exhibit life-long immunological memory. Combinations of cyclophosphamide plus TNF have analogous effects. In the E0771 breast tumor C57BL/6 mouse model, Dox plus TNF at doses which are without antitumor activity when given alone, cause complete cures of established tumors with concomitant stimulation of CTL and NK cells responses. The mechanisms involved in these therapeutic responses are discussed. In conclusion, the results obtained substantiate the possibility of establishing antitumor curative combination regimens based on the utilization of low non-toxic immunomodulating doses of certain anticancer drugs and specific cytokines.

A novel tumor necrosis factor-alpha inhibitory protein, TIP-B1.

TIP-B1, a novel TNF inhibitory protein, has been identified, purified and characterized from cytosolic extracts of TNF-treated human fibroblasts, and a partial TIP-B1 cDNA clone has been obtained. The (27 kDa pI approximately 4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids), and the nucleotide sequence of the corresponding cDNA clone. TNF-sensitive cells, when exposed to TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Thus, this TIP-B1 treatment effectively makes these cells TNF-resistant. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. TIP-B1 does not interfere with the interactions between TNF and the TNF receptors based on flow cytometric analysis of the cellular binding of biotinylated TNF. These and other data indicate that TIP-B1 is not a soluble TNF receptor, nor an anti-TNF antibody, nor a protease that degrades TNF, yet TIP-B1 functions when added exogenously to cells. Thus, TIP-B1 is not one of the proteins previously reported to be involved in resistance to TNF. The fact that incubation of the newly discovered novel TIP-B1 with TNF-sensitive cells protects them from TNF-induced cell death, including TNF-mediated apoptosis, makes TIP-B1 a candidate for therapeutic modulation of TNF-induced effects.

Identification, characterization, and cloning of TIP-B1, a novel protein inhibitor of tumor necrosis factor-induced lysis.

Some cancer cells evade elimination by virtue of their insensitivity to agents that induce apoptosis. Conversely, the side effects of anticancer agents could be diminished if normal cells were more resistant. To further elucidate the factors that contribute to the susceptibility of a cell to apoptosis, these investigations were designed to identify proteins isolated from cells exposed to low concentrations of tumor necrosis factor (TNF) that, when incubated with normally TNF-sensitive cells, protect these cells from TNF-induced cytotoxicity. TIP-B1, a novel protein, has been identified, purified, and characterized from cytosolic extracts of TNF-treated human fibroblasts. The approximately 27 kDa pI-4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids) and the nucleotide sequence of the corresponding 783-bp cDNA partial clone. Western blot analyses using polyclonal antisera raised against both the purified native TIP-B1 and the approximately 14 kDa product of the cDNA partial TIP-B1 clone, as well as Northern blot analyses using the cDNA insert as a probe, indicate that TIP-B1 may belong to a family of proteins that are expressed in a number of cell lines from diverse tissues. TNF-sensitive cells, when exposed to 4-10 microg/ml concentrations of TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. Preincubation of TIP-B1 with TNF does not affect the ability of TNF to induce lysis. Moreover, TIP-B1 does not seem to interfere with the interactions between TNF and the TNF receptors, based on a preliminary flow cytometric analysis of the cellular binding of biotinylated TNF. On the basis of these characteristics, TIP-B1 is not a soluble TNF receptor, an anti-TNF antibody, nor a protease that degrades TNF; yet TIP-B1 functions when added exogenously to cells. These characteristics, its novel sequence, and its function when added exogenously to cells indicate that TIP-B1 is unique and is not one of the other proteins reported previously to be involved in resistance to TNF. The ability of TIP-B1 to function after exogenous incubation with target cells makes TIP-B1 a likely candidate for therapeutic manipulation of TNF-induced effects.

Measurement of low-abundance cytokine mRNA in cells of murine lymphoid organs: a new quantitative reverse transcription/polymerase chain reaction method.

To investigate cytokine regulation in cells of freshly excised lymphoid tissues, rigorous quantitative reverse transcription/polymerase chain reaction (QRT-PCR) assays were developed to measure attomole (10(-18) mol) amounts of the mRNA for seven cytokines: interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), transforming growth factor beta (TGFbeta), IL-2 and IL-6. RNA was purified from single-cell suspensions of immune tissues (spleen, thymus and resident peritoneal cells). Data are presented demonstrating the utility of these assays for quantifying basal levels of all seven cytokine mRNAs in the freshly isolated splenocytes and thymocytes. Studies to establish the usefulness of these assays for measuring changes in the levels of cytokine mRNA focused on IL-1alpha, IL-1beta, TNFalpha and IL-2 in splenocytes, thymocytes and resident peritoneal cells. Using the QRT-PCR assays developed, levels of cytokine mRNA could be quantified in RNA samples obtained both from freshly isolated cells and from cells following short-term (

A novel TNF-inducible message with putative growth suppressor function.

We report the nucleotide sequence of a novel cDNA and TNF-induced expression of the corresponding message (mRNA) in human fibroblast cells. This message is also expressed in certain human tumor cell lines and is over-expressed in a colon cancer cell line (HT-29). NIH3T3 cells transfected with the antisense construct of the 5'-region of this novel cDNA formed 20-fold more colonies in culture compared to cells transfected with a sense construct of the same region or the sense and the antisense constructs of the central region of this cDNA. This observation suggests a possible growth suppressor function for the gene represented by this cDNA.

Thymic anti-tumor effectors in mice cured of lymphoma by cyclophosphamide plus TNF-alpha therapy: phenotypic and functional characterization up to 20 months after initial tumor inoculation.

As reported previously, cyclophosphamide plus tumor necrosis factor-alpha treatment of C57BL/6 mice bearing advanced EL4 lymphoma induced approx. 60% long-term (i.e., >60 days) survivors. These mice developed protective immunity, as evidenced by 1) rejection (100% survival) of EL4 tumor re-implanted on day 60 (day 0 = initial tumor implantation); and 2) development of significant levels of specific EL4 tumor cell killing activity by both splenocytes and thymocytes. Using this model, age-related changes in functionally and phenotypically definable thymocyte subsets were assessed. In thymocytes from 90 to 308 day survivors, specific immune memory was long term; both CD4+ and CD8+ cells were required for the ex vivo stimulation of lytic activity, but the specific anti-EL4 cytotoxic effector was CD4-CD8+. On day 520, the surviving mice were randomized into 2 groups. One group received a second re-challenge with EL4 tumor cells and all survived. The other group was sacrificed on day 520. Their thymocytes, exposed to X-irradiated EL4, developed anti-EL4 lytic activity and, in comparison with thymocytes of young and age-matched control mice, were markedly enriched in CD4-CD8+CD44+ cells. On day 625, thymocytes from the survivors of the day 520 re-challenge were evaluated and were found to have developed specific anti-EL4 lytic activity. Phenotypically, they had returned toward the pattern seen in age-matched control mice although CD4-CD8+CD44+ cells remained increased. These mice were > or = 2 years old, the median life span of C57BL/6 mice. Thus, mice cured of tumor by an immuno-modulating regimen rejected re-implanted primary tumor and maintained specific thymic anti-tumor immune memory for life.

Doxorubicin plus tumor necrosis factor alpha combination treatments in EL4-lymphoma-bearing C57BL/6 mice.

The therapeutic efficacy of a total of 42 single-agent or combination protocols involving doxorubicin (Adriamycin, ADM) and tumor necrosis factor alpha (TNFalpha) were evaluated in the syngeneic murine lymphoma model, C57BL/6-EL4. Combination treatments were the most effective and the therapeutic effects were schedule-dependent; e.g. it was generally advantageous for ADM to precede TNFalpha administration. Two protocols selected for further study were 4 mg/kg ADM i.v. on days 1 and 8 plus TNFalpha, i.v., at either 16000 U (7 microg)/injection, on days 1 and 8 or 4000 U (1.7 microg)/injection, on days 11-15. Survival of mice bearing one of four EL4 sublines having different in vitro drug sensitivities was assessed. These sublines were E10 (ADM-sensitive/TNFalpha-resistant), E16 (sensitive/sensitive), ER2 (ADM-resistant/TNFalpha-sensitive) and ER13 (resistant/resistant). Between 80% and 100% long-term survivors (i.e. tumor free on day 60) were obtained with the two treatments in mice bearing ADM-sensitive sublines, even though one of these sublines, E10, was resistant to TNFalpha in vitro. Induction of long-term survival appeared, therefore, to correlate with in vitro defined sensitivity/resistance to ADM, but not to TNFalpha Treatment-induced modulations of tumoricidal immune effector functions were also examined. Taken together, the results indicated that induction of long-term survival involved complex interactions of: (1) ADM-induced tumor modifications, including, but not limited to, tumor debulking, (2) combination-treatment-induced modifications of splenic cytolytic T cell and macrophage activities, and (3) the restoration of thymus cellularity. Finally, when long-term survivors resulting from treatment of E10- or E16-bearing mice were implanted with ER2 on day 120, the majority survived, indicating that long-term immune memory, capable of recognizing drug resistant variants, had been established.

Evidence for the involvement of ecto-5'-nucleotidase (CD73) in drug resistance.

Increased ecto-5'-nucleotidase (ecto-5'NT) protein expression in several multidrug-resistant (MDR) cell lines, documented previously by our group, suggests that this enzyme is involved in drug resistance. Here, Northern blot analysis of selected cell lines and their MDR variants positively correlated ecto-5'NT protein with its mRNA expression. An inhibitor of ecto-5'NT enzymatic activity, alpha,beta-methyleneadenosine 5'-diphosphate (AMP-CP), was used to determine if functionally active enzyme had a role in drug resistance. AMP-CP (0.3 mM) reversed the resistance of ecto-5'NT-positive MDR cells (MCF7/A6, L1210/A) to doxorubicin, whereas it did not affect the doxorubicin sensitivity of the ecto-5'NT-negative parental cell lines or that of 2 ecto-5'NT-negative MDR cell lines (HL60/VCR and A2780/DX5). Furthermore, AMP-CP increased rhodamine uptake and inhibited rhodamine efflux from ecto-5'NT-positive MDR cells without affecting ecto-5'NT-negative MDR cells. The presence of exogenous adenosine (0.5 microM) circumvented AMP-CP-induced inhibition of rhodamine efflux from EL4/ADM cells. AMP-CP inhibited the growth of the ecto-5'NT-positive L1210/A MDR cells but had no effect on the growth of the parental cell line. Determination of intracellular ATP levels indicated that MDR cells which had increased ecto-5'NT expression also had a lower intracellular ATP level than their parental cells. Our results suggest that, in certain MDR cell lines, ecto-5'NT serves as a required accessory molecule in resistance mediated by ATP-dependent mechanisms and that growth-sustaining nucleosides are provided by this salvage pathway.

Specific anti-EL4-lymphoma immunity in mice cured 2 years earlier with doxorubicin and interleukin-2.

This laboratory has reported the conditions for an effective, non-toxic, chemoimmunotherapy utilizing doxorubicin in combination with prolonged administration of interleukin-2 and the identification of the critical role of activated CD8+ T cells in the therapeutic effect. Mice (C57BL/6) cured in those studies have been followed for the remainder of their life spans. These mice, approximately 2 months of age when initially inoculated with syngeneic EL4 lymphoma, survived for more than 2 years, the normal life span of C57BL/6 mice. Mice 4 months old reinoculated with the EL4 cells all survived. At about 1 year of age mice were sacrificed and the ability of their thymocytes and splenocytes to develop specific CD8+ anti-EL4 activity was as high as it had been at the time of tumor rejection. At about 2 years of age EL4 was reimplanted into mice; all of them survived. These surviving mice, at 2 years 2 months of age, as well as a group of 2-year-old mice not rechallenged, were killed and functional antitumor activity and phenotype characteristics of various lymphocyte populations were determined in comparison to those of young and age-matched control mice. The phenotyping of the lymphocytes from the cured mice indicated very notable differences in subset distribution and increased CD44 expression. Functionally they developed high levels of anti-EL4 activity, which was ablated by combined treatment with monoclonal antibodies against CD8 and CD44, indicating the role of memory cells. Consistent with cells from aged mice, these same cell populations had a very reduced allogeneic responsiveness. It appears that cured mice have developed an immune memory specific for EL4.

Cyclophosphamide plus tumor necrosis factor-alpha chemoimmunotherapy cured mice: life-long immunity and rejection of re-implanted primary lymphoma.

Changes in functionally and phenotypically definable splenocyte subsets in aging mice which had been rendered tumor-free in early life by immunochemotherapy (cyclophosphamide plus tumor necrosis factor-alpha) were studied in the syngeneic EL4 lymphoma-C57BL/6 murine model. Treatment-induced long-term survivors (LTS) surviving rechallenge are termed "immune-LTS". On day 120 (day 0, initial tumor inoculation), splenocytes from day 60 rechallenged immune-LTS developed significantly greater specific anti-EL4 cytolytic activity in an ex vitro assay than those from non-rechallenged LTS. Splenocytes from combination-treated groups developed significantly higher activity than those from cyclophosphamide-induced immune-LTS. The splenic effector precursor was a CD8+ T cell. The specific anti-EL4 effector cell from the cyclophosphamide-induced immune-LTS was CD4- CD8+; however, approximately 50% of those from combination-treated immune-LTS appeared to be CD4+CD8+. On day 520 immune-LTS were randomized into 2 groups. One group was re-implanted with EL4 tumor; all mice survived. The other group was killed and, even though their splenocytes developed considerable anti-EL4 activity, their allogeneic responsiveness was as reduced as that of age-matched controls. Phenotypic analysis, compared with splenocytes from young and age-matched controls, revealed changes in the makeup of each T-cell subset, except the CD4+CD8+, and all subsets, except the CD4-CD8-, had increases in CD44 positivity. On day 625, the age of these mice was equivalent to the median life-span of C57BL/6 mice; nevertheless, their splenocytes developed high anti-EL4 activity. Phenotypic analysis indicated that, compared to day 520, there was a major decrease in CD4-CD8+ splenocytes; we suggest that these cells had migrated to the site of tumor eradication.

Protective specific immunity induced by cyclophosphamide plus tumor necrosis factor alpha combination treatment of EL4-lymphoma-bearing C57BL/6 mice.

A combination treatment protocol initiated 12 days after tumor injection, when the tumor was large, by administering cyclophosphamide (CY, 150 or 250 mg/kg) intraperitoneally followed by intravenous tumor necrosis factor alpha (TNF alpha, 1000 units injection) on days 13, 16, 18, 21, and 23, resulted in about 60% long-term survival (i.e., survival for at least 60 days) in the syngeneic C57BL/6 mouse/EL4 lymphoma model system. The establishment of a specific antitumor immune memory and its possible therapeutic relevance was verified by reinjecting 60-day survivors with EL4 cells; all 60-day survivors that had received the combination treatments rejected the implants and survived for a further 60 days. Thymic cellularity was reduced during treatment and its recovery appeared to correlate with long-term survival and immunity. Thymocytes from mice treated with the combination were found to express significant levels of specific anti-EL4 cytolytic activity following a 4-day stimulation culture with X-irradiated EL4 cells and low concentrations of interleukin-2. This response could not be generated with thymocytes from naive animals. In each case the effect seen with the combination of a moderate CY dose (150 mg/kg) with TNF alpha was better than that seen with either dose of CY alone and equal to or better than that seen with the higher dose of CY combined with TNF alpha. These results indicate that treatment with a single moderate dose of CY in combination with TNF alpha is effective against a large, established tumor in this murine model. Furthermore, all the long-term survivors induced by this treatment developed protective immunity against reimplanted tumor and demonstrated a long-term specific immune memory in the thymus.

Murine splenic macrophage tumoricidal activation by cytokines.

Interleukin-2 (IL-2), IL-1 beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha), each alone and in all possible combinations, were studied for their capacity to activate murine resident splenic macrophages to a tumoricidal state. Two approaches were used in these studies. The first approach was to add cytokine directly to the adherent macrophages that had been washed free of nonadherent spleen cells. The only agent effective alone was IL-2, inducing significant tumoricidal activity in macrophages obtained after culturing whole spleen cell suspensions for 4, but not 1 to 3, days. Nonadherent splenic populations were required during this 4-day macrophage "culture conditioning." Only combinations of cytokines containing IL-2 were effective, but none more than IL-2 alone. The second approach was to add cytokine to the whole spleen cell suspensions for an activation period before isolation of adherent macrophages. Again, the only agent effective alone was IL-2. Macrophage tumoricidal activity was highest when IL-2 was added to the whole spleen cell suspensions at the initiation of the 4-day activation culture. In addition, TNF-alpha, but none of the other cytokines, significantly augmented the IL-2-induced effect. The tumoricidal activity was not a consequence of lipopolysaccharide contamination or of lymphokine-activated killer cells. Based on the utilization of neutralizing antibodies, IL-1 alpha, IL-1 beta, and IFN-gamma were not involved as soluble mediators during the activation of tumoricidal splenic macrophages by IL-2 with or without TNF-alpha.

Doxorubicin and cyclosporin A affect murine lymphoid cells expressing different antigenic determinants.

Cyclosporin A (CsA) enhances the antitumor activity of doxorubicin (Dox) as well as that of some other cytotoxic drugs against drug-resistant tumor variants. In some cases, however, such combination treatments result in severe unexplained toxicities. In this study the possibility was explored that the effects of CsA and Dox on lymphoid cells may be instrumental, at least in part, in determining their toxicological effects. Subsets of spleen and thymus lymphoid cells, from mice with or without Dox or CsA treatment, were identified by flow cytometry based upon their plasma membrane antigenic determinants. The results indicate that there is essentially no cross-sensitivity/resistance between the two agents. The most Dox-susceptible cells were immature (non-proliferating) CD4+CD8+ thymocytes and CD3-B220- as well as CD3-B220+ splenocytes. These populations were intact following CsA treatment, but the numbers of mature CD4+CD8- and CD4-CD8+ cells were substantially reduced. Similar "mirror image" differences were found for other populations examined. When considered together, these findings suggest that in combination Dox and CsA would affect nearly all subsets of lymphoid cells, providing one possible explanation of why increased leukopenia, toxicity and immunosuppression are found after their combined administration. Since leukemias, lymphomas and, to a more limited extent, certain solid tumors express these same phenotypic markers, similar analyses should be considered for monitoring and perhaps even predicting neoplastic cell sensitivity to treatment with such agents.

Lipopolysaccharide and splenic tumoricidal macrophage activation.

Splenic macrophage tumoricidal activity was examined and a splenic macrophage tumoricidal assay was established. Initially, mixtures of lipopolysaccharide (LPS) and spleen single cell suspensions (SSCS) were cultured for 1-4 days. Adherent macrophages, washed free of nonadherent cells and LPS, were then examined and were found to lack tumoricidal activity in a standard 18-h 51Cr release assay. However, tumoricidal activity was generated if LPS was added to the SSCS cultures at later time points during the 4-day incubation period; maximal activity was seen when LPS was added on day 3. In parallel, significant changes in macrophage autofluorescence and morphology, but not phenotype, were observed. Next, SSCS were cultured for 1-4 days without stimulating agents. Adherent macrophages were then washed free of nonadherent cells and LPS was added. Significant tumoricidal activity developed in time- and LPS concentration-dependent fashions. The presence of nonadherent spleen cells in physical contact with the macrophages during the SSCS culture was essential for the macrophages in the resultant monolayer to be responsive to LPS. Activated splenic macrophage-mediated lysis of tumor cells was shown to depend on the contact between the two cells.

Doxorubicin-induced DNA degradation in murine thymocytes.

Exposure of murine thymocytes to doxorubicin (Dox) (0.5-1.0 microM, 24 hr) triggered rapid DNA degradation, as indicated by the appearance of a major subdiploid population demonstrated by DNA flow cytometry. Electron microscopic comparison of samples with large subdiploid populations versus those with little or no such subset revealed significantly more cells with the characteristic features of apoptosis, the morphologically definable stage of programmed cell death. These features include unipolar condensed chromatin, zeiosis, and electron-dense cytoplasm. Dox-induced apoptosis occurred without prior S or G2/M phase arrest or cell size increase. The subset most susceptible to Dox-induced apoptosis in vitro and in vivo was CD3-CD4+CD8+. The same subset is affected by dexamethasone (Dex); as reported for Dex-induced apoptosis, actinomycin D and cycloheximide also blocked Dox-induced apoptosis. Thymocytes exposed to higher Dox concentrations (2-10 microM) did not have a subdiploid population. Although at 2-10 microM Dox significantly reduced cell numbers (probably as a result of necrosis), at least 5-10% of the population was viable at 24 hr. Thymocytes exposed to low concentrations of Dox (0.001-1.0 microM) plus Dex (0.1 microM) exhibited additive induction of apoptosis, whereas those exposed to high concentrations of Dox (2-10 microM) plus Dex were completely devoid of any evidence of apoptosis. These results indicate that the Dox-induced killing in thymocytes (mostly noncycling cells) occurs via different mechanisms depending upon the Dox concentration.

Ecto-5'-nucleotidase (CD73) in multidrug-resistant cell lines generated by doxorubicin.

Cytochemical screening for a panel of enzymes revealed increased 5' nucleotidase (5'NT) expression in 3 of 3 P-glycoprotein 170 (Pgp170)-positive multidrug-resistant (MDR) variants of the murine EL4 T-lymphoma cell line (EL4/ADM, ER2 and ER13). Electron microscopic localization established the presence of the membrane-bound ecto-form of the enzyme. Nine other murine, human and Chinese hamster cell lines and their MDR variants were tested for ecto-5'NT. Of these, 4 MDR variants (human cell lines MCF7A6, MCF7A2, HeLaJ2C and the murine cell line L1210A) showed increased expression of ecto-5'NT, when compared with their parental cell lines. The findings with cells of human origin were confirmed by immunofluorescent localization with a specific monoclonal antibody (MAb) (27.2) against the human ecto-5'NT. All MDR cell lines with elevated ecto-5'NT expression were generated by doxorubicin treatment. These cells were more sensitive than their parental cell lines to AMP at concentrations of 1.5-3.0 mM, confirming that the expressed ecto-5'NT was biologically active. The parental and MDR cells did not differ, in general, in their sensitivity to adenosine. An inhibitor of ecto-5'NT, alpha,beta-methyleneadenosine 5'-diphosphate, completely reversed the resistance of the EL4/ADM cell line to doxorubicin. The possibility exists of a functional relationship between the ecto-5'NT molecule and the members of the ATP-binding cassette transporter superfamily, important components of MDR, in some cell types.

Effect of perioperative chemoimmunotherapy with cyclophosphamide and autologous tumor vaccine in murine MBT-2 bladder cancer.

The in vitro cytotoxic activity of splenocytes from C3H/He mice implanted subcutaneously with 10(6) syngeneic MBT-2 tumor cells on day 0 was significantly enhanced after cyclophosphamide (100 mg./kg., intraperitoneally) given 2 days before tumor resection on day 17, with or without active specific immunization with BCG plus autologous irradiated tumor cells (vaccine) 1 week after tumor resection. Furthermore, a significantly lower tumor incidence was seen in mice challenged with 10(5), but not 10(6), tumor cells per mouse 24 hours after tumor resection on day 17 and treated with cyclophosphamide on day 15 and postoperatively with vaccine than was found in nontreated tumor resected mice. Phenotypic analysis of cells from spleen showed that cyclophosphamide pretreatment and postoperative vaccine, either singly or in combination, induced a significant increase of both CD44+ memory T cells and CD11b+ myeloid/macrophage cells. Thus, in addition to a specific antitumor immune response, a nonspecific cytolytic mechanism may also play a role in the observed antitumor effect.