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Melanoma antigen family A4 (MAGEA4), a cancer/testis antigen, is overexpressed and is thus an immunotherapy target in various malignant tumors, including non-small cell lung cancer. However, whether MAGEA4 induces or inhibits the apoptosis of lung cancer cells remains controversial, as is its prognostic significance, particularly since there is no reliable method with which to detect MAGEA4 specifically. In this study, we optimized assay conditions to detect MAGEA4 based on cells transiently transfected with MAGEA genes, and found that MAGEA4 was expressed in four of eight non-small cell lung cancer cell lines, and in 25.4% of clinical lung cancer specimens. We also found that MAGEA4 overexpression decreased apoptosis, as measured by the levels of cleaved caspase-3 in stably transfected 293F cells. Notably, patients with nuclear MAGEA4, but not p53 expression exhibited a significantly poorer survival than those expressing both nuclear MAGEA4 and p53. Indeed, multivariate analysis identified nuclear MAGEA4 as an independent prognostic factor (P=0.0042), albeit only in the absence of p53. In this study, to the best of our knowledge, we are the first to demonstrate that the function and prognostic value of MAGEA4 depends on its subcellular localization and on the p53 status.

Expression of src related proto-oncogenes in non-Hodgkin's lymphomas and non-malignant lymph nodes was analyzed by means of in situ hybridization assays with biotinylated DNA probes. In 36 cases of non-Hodgkin's lymphomas, both c-mos and c-abl were expressed in 27 cases, and c-erbB and c-src were expressed in 15 cases and 7 cases, respectively. No case expressed c-fps or c-yes. On the contrary, in 11 cases of non-malignant lymph nodes c-erbB and c-mos was expressed in only 3 cases. No other proto-oncogenes were expressed. Lymphomas in general express multiple proto-oncogenes simultaneously. A variety of combinations of expressed proto-oncogenes were observed suggesting diversity among non-Hodgkin's lymphomas in biological characteristics. However, a clear association with the expression of a single proto-oncogene or the number of expressed proto-oncogenes with T cell / B cell types, or the histopathological classification of NHL, or disease prognosis was not observed.

We analyzed the genetic origins of anti-GD3 antibodies by comparing nucleotide sequences of the variable regions from the human monoclonal antibody (mAb), 27-26 (mu, k), established from a patient with leukemia, and another human anti-GD3 mAb, HJM-1 (mu, lambda) derived from a patient with melanoma. The variable regions of 27-26 and HJM-1 were remarkably similar to the germ-line genes. The mAb 27-26 was thought to be derived from germ-line repertoire expanded throughout our experiment. HJM-1 was derived from lymphocytes stimulated by GD3 abundantly expressed on melanoma cells.

Peripheral blood mononulear cells (PBMC) derived from 10 asymptomatic human T lymphotropic virus type I (HTLV-I) carriers were cultured for a short term (10-14 days) in the absence of exogenous antigens. In 5 carriers, when compared with 5 HTLV-I non-carriers an apparent increase in the proportion of CD8+ DR+ cells was observed. The clonality of cultured lymphocytes was then examined by analyzing the usage of Vbeta families of T cell receptor genes. In three of the 5 carriers with an increased CD8+ population, two to four Vbeta genes were dominant in the CD8+ population but not in the CD4+ population. No dominance of Vbeta gene usage was observed in lymphocytes derived from the 5 noncarriers. The sequence of cDNA from Vbeta families which were especially dominant revealed their oligoclonal characteristics. These results were quite similar to our previous findings from HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients in whom the same oligoclonal CD8+ cells were expanded cytotoxic T lymphocyte clones for HTLV-I genome products. We posed the question of whether the dominance of TCR Vbeta usage in cultured PBMC was associated with the HTLV-I genome dose in the PBMC or with anti-HTLV-I antibody titers. The three carriers who showed an increased CD8+ population mentioned above all showed a rather high HTLV-I genome dose, which again was similar to HAM/TSP patients. These three carriers however, did not necessarily show high anti-HTLV-I antibody titers in contrast with HAM/TSP patients, who generally do.

Receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) share a common beta subunit. We recently reported that GM-CSF acts in concert with transforming growth factor-beta1 (TGF-beta1) and Notch ligand Delta-1 (Delta-1) to promote the differentiation of human blood monocytes into Langerhans cells. In the present study, we examined whether IL-3, in place of GM-CSF, can induce the development of Langerhans cells from blood monocytes in the presence of TGF-beta1 and Delta-1, because the IL-3 receptor alpha chain was substantially expressed on monocytes. However, the generation of Langerhans cells was not obtained by the combination of IL-3, TGF-beta1 and Delta-1, even though GM-CSF and IL-3 exhibited a similar effect with respect to the differentiation of monocytes into macrophages and dendritic cells. The addition of GM-CSF to the culture supplemented with IL-3, TGF-beta1 and Delta-1 restored the differentiation of monocytes toward Langerhans cells. A microarray analysis revealed that a number of genes including Langerhans cell markers, E-cadherin and Langerin, were specifically expressed in cells from GM-CSF-containing cultures but not in those from IL-3-containing cultures. These data suggest that IL-3 can not replace GM-CSF to induce the differentiation of human monocytes into Langerhans cells in culture.

Histone acetylation controls the expression of specific genes in eukaryotic cells. We investigated the role of histone deacetylases (HDACs) in the differentiation of human erythroid cells, using pharmacological approaches. When CD36+ erythroid precursor cells, generated from CD34+ cells with stem cell factor, flt-3 ligand, thrombopoietin, interleukin-3, interleukin-6, and erythropoietin, were cultured with an HDAC inhibitor FK228 (depsipeptide) at a specified dose in the presence of erythropoietin, their differentiation was inhibited, as determined by the expression of CD45 and glycophorin A. Addition of the same dose of FK228 to cultures did not affect the growth of CD36+ cells. Regardless of the presence or absence of FK228, cultured CD36+ cells displayed similar proliferation kinetics. Analysis of acetylated histones revealed that FK228 upregulated the acetylation status of histones H3 and H4 in CD36+ cells. The inhibition of CD36+ cell differentiation was restored by removal of FK228 from the culture, indicating that the modification of CD36+ cell differentiation by FK228 is reversible. Furthermore, interference with histone deacetylation by FK228 inhibited the generation of CD36+ erythroid cells from CD34+ hematopoietic progenitor cells. Our results indicate the possible involvement of HDACs in human erythropoiesis, especially the regulation of erythroid cell differentiation.

Interleukin (IL)-4 and IL-10 have a wide variety of activities in the immune system. We re-evaluated the action of IL-4 and IL-10 on human blood monocytes, myeloid dendritic cell (DC1) precursors, using a serum-free culture system. Both IL-4 and IL-10 inhibited the survival of CD14 + monocytes supported by granulocyte-macrophage colony-stimulating factor in a dose-dependent manner. When IL-4 and IL-10 were combined, they had synergistic effects at low doses and induced a profound suppression of CD14 + monocyte survival. When the optimal timing was determined, the exposure to IL-4 and IL-10 for the initial 2 days was essential for suppression of survival of CD14 + monocytes. Annexin V/propidium iodide staining indicates that the suppression of CD14 + monocyte survival induced by IL-4 and IL-10 results from apoptosis. Tumor necrosis factor-a and lipopolysaccharide abrogated the effects of IL-4 and IL-10 on CD14 + monocytes, albeit incompletely. Thus, IL-4 in synergy with IL-10 negatively regulates the survival of DC1 precursor monocytes by inducing their apoptosis, which is modulated by factors such as tumor necrosis factor-a and lipopolysaccharide. Our data suggest the primary activities of IL-4 and IL-10 in DC1-mediated immune responses.

Acute myeloid leukemia (AML) has distinct subgroups characterized by different maturation and specific chromosomal translocation. In order to gain insight into the gene expression activities in AML, we carried out a gene expression profiling study with 21 AML samples using cDNA microarrays, focusing on acute promyelocytic leukemia with specific translocation t(15;17)(q22;q12) [French-American-British or FAB-M3 with t(15;17)] and AML without maturation (FAB-M1) characterized by morphologically and phenotypically immature AML blasts and no recurrent chromosomal abnormalities. Using a multivariate sigma-classifier algorithm, we identified 33 strong feature genes that distinguish FAB-M3 with t(15;17) from other AML samples, and 24 strong feature genes that classify FAB-M1. A direct comparison between FAB-M3 with t(15;17) and FAB-M1 led to selection of 13 strong feature genes. Those genes include some known to be related to leukemogenesis and cell differentiation. RIN1, a gene in the ras pathway, was up-regulated in FAB-M3 with t(15;17). Growth factor-binding protein 2 gene was down-regulated in FAB-M1. Huntingtin gene was up-regulated in FAB-M1. Others include syndecan 4, interleukin-2 receptor beta, folate receptor beta, low affinity immunoglobulin gamma, Fc receptor IIC precursor, insulin-like growth factor binding protein 2, and myeloperoxidase, which are involved in cell differentiation. Overexpression of myeloperoxidase in FAB-M3 cells with t(15;17) compared to FAB-M1 cells is consistent with the conventional cytochemical staining pattern. Thus, the study revealed that a morphologically-defined FAB-M1 subtype has a distinct gene expression signature that contributes to its cell differentiation and proliferation as well as FAB-M3 with a recurrent cytogenetic abnormality t(15;17)(q22;q12).

Acute myeloid leukemia (AML) has distinct subgroups characterized by different maturation and specific chromosomal translocation. In order to gain insight into the gene expression activities in AML, we carried out a gene expression profiling study with 21 AML samples using cDNA microarrays, focusing on acute promyelocytic leukemia with specific translocation t(15;17)(q22;q12) [French-American-British or FAB-M3 with t(15;17)] and AML without maturation (FAB-M1) characterized by morphologically and phenotypically immature AML blasts and no recurrent chromosomal abnormalities. Using a multivariate sigma-classifier algorithm, we identified 33 strong feature genes that distinguish FAB-M3 with t(15;17) from other AML samples, and 24 strong feature genes that classify FAB-M1. A direct comparison between FAB-M3 with t(15;17) and FAB-M1 led to selection of 13 strong feature genes. Those genes include some known to be related to leukemogenesis and cell differentiation. RIN1, a gene in the ras pathway, was up-regulated in FAB-M3 with t(15;17). Growth factor-binding protein 2 gene was down-regulated in FAB-M1. Huntingtin gene was up-regulated in FAB-M1. Others include syndecan 4, interleukin-2 receptor beta, folate receptor beta, low affinity immunoglobulin gamma, Fc receptor IIC precursor, insulin-like growth factor binding protein 2, and myeloperoxidase, which are involved in cell differentiation. Overexpression of myeloperoxidase in FAB-M3 cells with t(15;17) compared to FAB-M1 cells is consistent with the conventional cytochemical staining pattern. Thus, the study revealed that a morphologically-defined FAB-M1 subtype has a distinct gene expression signature that contributes to its cell differentiation and proliferation as well as FAB-M3 with a recurrent cytogenetic abnormality t(15;17)(q22;q12).

In this study, we analyzed specific anti-tumor immune responses in tumor-bearing hosts by measuring HER2-specific CD8 + T cell responses. No measurable HER2-derived peptide (HER2p63)-specific CD8 + T cells were present in the spleens of mice in the early to late phase of tumor-bearing. Vaccination with HER2 protein and cholesteryl group-bearing pullulan (CHP-HER2 complex) induced HER2-specific CD8 + T cells, but their numbers continuously declined as tumors continued growing. Removal of CD4 + T cells by anti-CD4 monoclonal antibody in the early tumor-bearing stage resulted in tumor regression. The combination of CHP-HER2 complex vaccination and depletion of CD4 + T cells enhanced and restored HER2-specific CD8 + T cells in the late stage of tumor-bearing, and also suppressed tumor growth. These results indicate the importance of manipulation of CD4 + T cells in developing effective immunotherapies as cancer vaccines.

T lymphocytes and NK cells play an important role in the graft-versus-host (GVH) disease. IL-12 is known to activate both lymphocyte subpopulations. Here, we examined the effect of IL-12 on the outcome after allogenic bone marrow transplantation (allo-BMT). Experiments were performed in a major histocompatibility complex (MHC)-matched allo-BMT model from DBA/2 to BALB/c mice. We administered IL-12 subcutaneously to recipient BALB/c mice at the day of BMT and 2 days later. At a dose of 200 ng IL-12, mice died between day 4 and day 10. When 50 ng of IL-12 was administered, 50% of the animals died, at a dose of 25 ng all mice recovered. In vivo blocking experiments with antagonistic monoclonal antibodies indicated that the early lethality was mediated by FasL, IFNgamma and TNFalpha. Recognition of third party antigens by donor, but not recipient lymphocytes caused lethality. HE staining of the large intestine of IL-12 treated animals showed a severe mucosal injury associated with infiltrating lymphocytes and apoptotic bodies. In IL-12 treated mice in contrast to untreated control animals, flow cytometry analysis revealed numerous CD3+ T cells and CD11c+ dendritic cells (DC) were found in the large intestine. The interaction of T cells and antigen presenting DC might contribute to the lethality in our system. In light of these findings, the usefulness of IL-12 application for patients after BMT is rather questionable.

It has been reported that expression of Bax by Tet-On system induces apoptosis in Jurkat cells. The parental Jurkat cells have mutation of Bax gene and do not express Bax protein. Wild-type Bax-bearing cells express endogenous Bax protein and it is still unclear whether overexpression of Bax alone can sufficiently induce apoptosis in these cells in the absence of any cytotoxic stimulus. To investigate this, wild-type Bax-bearing K562 cells were transfected with Tet-On Bax-inducible system (pTet-On and pTRE-Bax plasmids), and Bax-inducible stable cell lines were established. Overexpression of Bax in wild-type Bax-bearing K562 cells without any cyctotoxic signal resulted in increase of apoptotic cells, caspase-3 activation, mitochondrial release of cytochrome c, and mitochondrial membrane potential change. Western blotting and confocal microscopy revealed that overexpression of Bax was detected in mitochondria. A pancaspase inhibitor, zVAD-fmk, which has no effect on mitochondrial cytochrome c release and mitochondrial membrane potential change inhibited the apoptotic events in the presence of overexpressed Bax in mitochondria. These findings suggest that Bax protein, when present above a threshold level, is sufficient to trigger an apoptosis cascade, and its initiation requires simultaneous caspase activation probably not mediated by mitochondrial cytochrome c release and mitochondrial membrane potential change in K562 cells.

We have previously shown that protein phosphatase-1 (PP1) is the most abundant Ser/Thr phosphatase in human adult primary leukemic cells. To determine the clinical importance of PP1 expression, we compared PP1 activity of leukemic blasts with other putative prognostic factors in 46 patients with acute myelogenous leukemia (AML) who were treated with remission induction chemotherapy. PP1 was ubiquitously but differently expressed in various FAB subtypes (M1-M5), although PP1 activity was significantly higher in blasts of AML-M4 than in AML-M2. PP1 activity was significantly lower in elderly patients > or =55 years (P=0.005), and in those with high white cell counts > or =100,000/microl (P=0.039) at initial diagnosis. Correlation was observed between PP1 activity ( or =0.15 nmol/min/10(8) cells) and prognosis of AML patients. Eleven of 46 patients with less than 0.15 nmol/min/10(8) cells (low PP1 activity group) had significantly lower overall survival than those with > or =0.15 nmol/min/10(8) cells (high PP1 activity group). The median overall survival was 8 months for patients with low PP1 activity compared to 27 months for those with high PP1 activity in their AML cells. Multivariate analysis using Cox's proportional hazard model showed that low PP1 activity significantly contributed to prognosis. This preliminary study suggests that low PP1 activity may be associated with shortened survival time for AML patients with high white cell counts.

Gangliosides have been thought to be tumor markers of various neuroectoderm-derived cancers. However, their roles in malignant phenotypes of cancer cells are not well understood. We have performed the remodeling of ganglioside profiles of mouse melanoma B16 (B78 subline) by introducing GM2/GD2 synthase cDNA, and analyzed the phenotypic changes such as cell morphology, growth, adhesion and c-fos gene expression. Although newly expressed GM2 was clearly detected, GM2-positive transfectant cells showed rather reduced growth rates in vivo and in vitro, lower expression levels of c-fos gene and increased levels of cell adhesion to extracellular matrices compared to control cells. These results suggested that GM2 is involved in the regulation of cell-cell or cell-extracellular matrix interaction, and negatively control cell proliferation.

To evaluate whether overexpression of Bax, an apoptosis-promoting gene, sensitizes KATOIII gastric cancer cells to apoptosis induced by chemotherapeutic agents, three stable cell lines of KATOIII transfected with Bax (KATOIII-Bax), Bcl-2 (KATOIII-Bcl-2), or control pCI-neo expression vector (KATOIII-pCI-neo) were established. The cells were treated with paclitaxel, 5-fluorouracil, or doxorubicin, and the apoptotic response was measured. Our results showed that the sensitivity of the KATOIII-Bax cells to chemotherapeutic agents was enhanced compared with that of the KATOIII-pCI-neo cells, and the KATOIII-Bcl-2 cells were more resistant to these agents. Western blotting revealed that cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells. Significant increase of cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was detected 24 h after exposure to chemotherapeutic agents, when apoptotic cells were less than 10%. The cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells at all time points examined after exposure to chemotherapeutic agents. Marked activation of caspase-3 in the KATOIII-Bax cells was observed 48 h and 72 h after exposure to chemotherapeutic agents compared with that in the KATOIII-pCI-neo cells. Consistently, zVAD-fmk, a pancaspase inhibitor, repressed the paclitaxel-induced apoptosis. In addition, Bcl-2 overexpression strongly blocked KATOIII cell apoptosis by inhibiting the cytochrome c release from mitochondria and caspase-3 activation. These findings suggest that cytochrome c release is a major mechanism of apoptotic response and Bax overexpression sensitizes KATOIII cells to chemotherapeutic agent-induced apoptosis through enhancing the release of cytochrome c from mitochondria.

The complement system of the human body inactivates the infectious ability of retroviruses injected as an artificial gene transfer vector. We established new murine leukemia virus (MuLV) packaging cell lines; D2SS and D7S which express decay-accelerating factor (DAF) on their surface. Both D2SS and D7S were resistant against incubation with fresh human serum. Moreover, the retroviruses produced from these packaging cell lines were also resistant to serum treatment. This resistance can be inhibited by DAF neutralizing antibody 1C6. These data demostrate that DAF induces a partial protection of MuLV infection from the human complement system.

We have previously shown that a novel hydrophobized polysaccharide/oncoprotein complex vaccine can induce immune responses against the HER2/neu/c-erbB2 (HER2) expressing tumors. Bone marrow-derived dendritic cells (DCs), as antigen presenting cells (APCs), are the first candidates for presentation of tumor antigens. The aim of this study was to see whether DCs are able to elicit antigen specific host immune responses by stimulating the proliferation of T cells after exposure to cholesteryl group bearing pullulan (CHP) and HER2 protein complex. Vaccination by CHP-HER2 complex was as effective as cholesteryl group bearing mannan (CHM) and HER2 complex on which we reported previously. Immunization of mice with HER2 expressing CMS17HE tumor cells generated both CD4+ T cells and CD8+ T cells reactive with CHP-HER2 complex pretreated DCs. In addition, immunization with either CHP-HER2 complex or HER2 protein alone could also generate both CD4+ T cells and CD8+ T cells specifically reactive with CHP-HER2 complex pretreated DCs. The complete rejection of tumors occurred when immunization with CHP-HER2 complex pretreated DCs was started 10 days after tumor inoculation. Therefore, bone marrow-derived DCs pretreated with hydrophobized polysaccharide/oncoprotein complex are a powerful tool for enhancing the effectiveness of oncoprotein for anti-tumor vaccination, opening new options for immune cell therapy.

The immunological screening of a cDNA phage expression library prepared from an adult T cell leukemia (ATL) cell line, ST1, was performed with IgG class antibodies in the serum of an ATL patient by the SEREX method to analyze the repertoire of antigen molecules in ATL. Ten different cDNAs, 7 previously reported and 3 newly identified, were isolated. One of the identified cDNAs was homologous to the recently reported gene, HTLV-I U5RE binding protein 1 (HUB1) encoding a protein binding to a possible repressor element of the long terminal repeat of the human T lymphotropic virus type I (HTLV-I). The obtained cDNA was expressed as a fusion protein with glutathione S-transferase. HUB1 protein was prepared by subsequent treatment of the fusion protein with thrombin. Reactivity of IgG serum samples from ATL patients, asymptomatic HTLV-I carriers and normal donors against the purified protein was examined by Western blot analysis. Twenty-one out of 30 samples (70.0%) from ATL patients, 10/24 samples (41.7%) from HTLV-I carriers and 9/24 samples (37.5%) from healthy donors showed positive reactivity, indicating the autoantigenicity of HUB1. The development of ATL may be related to higher production of antibodies against HUB1.

We examined the effects of interleukin (IL)-13 and IL-4 on growth of hematopoietic progenitors from 5-fluorouracil (5-FU)-treated mice, using an in vitro culture system. IL-13 or IL-4 alone failed to support colony formation by enriched marrow cells. IL-4 but not IL-13 in combination with IL-11 yielded a significant number of colonies. Neither IL-4 nor IL-13 affected colony formation supported by IL-3. When tested with two-factor combinations, IL-4 but not IL-13 suppressed the formation of colonies including multilineage colonies in the absence of IL-11. The inhibitory effects of IL-4 were not lineage-specific. Delayed addition experiments demonstrated that IL-4 is inhibitory in the early stage of colony formation. Effects of IL-4 on colony formation by pooled blast cells derived from 5-day culture of post-5-FU marrow cells was not evident. These findings indicate that IL-4 but not IL-13 is a negative regulator of hematopoietic progenitors, suggesting distinctive roles for IL-4 and IL-13 in early hematopoiesis.

Differentiation of leukemic HL-60 cells by all transretinoic acid (ATRA) resulted in a reduced rate of growth. Cyclosporin A and FK506, at concentrations that inhibited calcineurin activity, abrogated the ATRA-induced inhibition of HL-60 cell growth but these immunosuppressants had no effect on the ATRA-induced granulocytic differentiation. Treatment with 1 microM ATRA led to a progressive increase in calcineurin phosphatase activity of HL-60 cells; the increase in this activity appeared to parallel the functional change of HL-60 cells during granulocytic differentiation. Increase in calcineurin activity was concordant with the increased expressions of calcineurin A and calcineurin B subunit proteins. The FKBP12 expression increased during ATRA-induced differentiation and expression of cyclophilin A remained unchanged. We propose that the increased expression of calcineurin is involved in the ATRA-induced inhibition of HL-60 cell proliferation, as in the case with 1,25alpha-dihydroxy-vitamin D3.

In most normal somatic cells the telomeres of human chromosomes shorten with each cell division because of low expression or lack of telomerase activity. Telomerase, a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends, is reactivated or upregulated in tumor cells and maintains the stability of telomere length. We previously showed that treatment of HL60 leukemia cells with differentiation-inducing agents resulted in inhibition of telomerase activity. In the present study, we found that the decrease in telomerase activity did not temporally correlate with the expression of a differentiation marker, CD11b, on the cell surface. Mixing of protein extracts from telomerase-negative differentiated HL60 cells with those from parental HL60 cells did not result in inhibition of telomerase activity, suggesting that a diffusible cellular telomerase inhibitor was not produced in the differentiated cells. However, a decrease in telomerase activity correlated with a selective decrease of telomerase RNA expression, a decrease in the levels of total cellular RNA, and an increase in cells at G(0) phase.

The involvement of protein Ser/Thr phosphatase types 2A (PP2A) and 1 (PP1) in tumor necrosis factor alpha (TNF alpha)-induced tissue factor (TF) expression of human umbilical vein endothelial cells (HUVEC) was investigated. PP2A was more abundant than PP1 in the cytosol of HUVEC. CAL-A (0.5 nM) and OKA (2 nM), cell permeable inhibitors of PP1 and PP2A, augmented TNF alpha-induced TF expression, although TF expression induced by either TPA or thrombin was unchanged in the presence of GAL-A. Addition of CAL-A (0.5 nM) to TNF alpha-stimulated cultures led to an increase in the accumulation of TF transcripts. CAL-A (0.5 nM) also augmented the TNF alpha-induced phosphorylation of I kappa B-alpha, an inhibitor of NF-kappa B. Since PP2A is more sensitive to OKA than PP1, these results suggest that PP2A may be involved in regulating TNF alpha-induced TF expression in HUVEC through I kappa B-alpha dephosphorylation.

Stromal cells isolated from lymph nodes of adult T-cell leukemia/lymphoma (ATL) patients were cultured. Such lymph node stromal cells (LNSC) could be maintained for more than one year, whereas LNSC from other lymphoproliferative disorders ceased to proliferate within months. The rate of human T lymphotropic virus type I (HTLV-I) integration in these LNSC was examined by nested polymerase chain reaction (PCR) and estimated to be about 1 genome per 100 cells. These LNSC showed the same combination of cytokine production irrespective of the patient origin, granulocyte-macrophage (GM)-CSF, G-CSF, interleukin (IL)-1 beta, IL-6, interferon (IFN)-gamma and IL-8, being positive but not M-CSF, IL-1 alpha, IFN-alpha, tumor necrosis factor (TNF)-alpha, IL-2, LD78 and the IL-1 receptor antagonist (IL-1ra). The results show that LNSC from ATL patients have pronounced proliferation activity and constitutively secrete various cytokines. They therefore provide useful models for studying the microenvironment of lymph nodes in vitro, and especially the growth mechanism of ATL cells.

A murine monoclonal antibody, S2-566, was generated by immunization with a melanoma line, SK-MEL-28. Although AbS2-566 showed a similar reaction pattern with that of anti-GD3 mAbR24 in cell surface serology, it recognized a disialyl galactosyl structure not only in GD3 but in GT1a and GQ1b. The suppression of the growth of cultured melanoma cells by AbS2-566 was not obvious, in contrast with AbR24. However, the mitogenic effects of AbS2-566 on peripheral lymphocytes were observed showing similar kinetics to those of mAbR24, suggesting different signaling effects based on the different epitope structures.

The relevance of mutated p53 to in vivo rejection of MC-induced fibrosarcomas and/or in vitro CTL activity against these tumors was investigated. p53 gene was found to be altered in nine MC-induced fibrosarcomas of BALB/c origin. The mutated p53 cDNA derived from several MC-induced fibrosarcomas was transduced into CMS8 which lacks p53 expression. Immunization of mice with these mutated p53 transfectants failed to protect them from original MC-induced fibrosarcomas from which mutated p53 genes were derived. CTL lines specific for these MC-induced fibrosarcomas destroyed the original MC-induced fibrosarcomas, but not CMS8 transduced with mutated p53 genes derived from the same lines. A series of 9mer peptides consisting of mutated amino acid residues of p53 of Meth A, CMS17 and CMS9 were prepared, and target P1HTR cells were pulsed with them. None of CTLs for these fibrosarcomas were reactive with P1HTR pulsed with these peptides. In conclusion, peptides derived from mutated p53 genes may serve as target antigens for CD8(+) MHC class I restricted CTL as reported, but may often not contribute as target antigens to the rejection of MC-induced fibrosarcomas.

Using a fusion protein encoded by variant exon (v) 8-10 of CD44, a monoclonal antibody (mAb) 44-1V reactive with v9 product was generated. Western immunoblots and reverse transcriptase-polymerase chain reaction/Southern blot revealed that a 130 kilodalton component containing v8-10 products was specifically expressed in colorectal and gastric cancer cell lines. Immunohistological examination of 179 pairs of colorectal cancer and normal tissues revealed that about 47% of cancers expressed the CD44 variant (CD44v8-10), and its expression correlated with hematogenous and lymph node metastasis. These findings suggest the usefulness of CD44v8-10 in the prediction of colorectal cancer metastasis.

We examined the effects of granulocyte colony-stimulating factor (G-CSF) on cell-cycling of hematopoietic progenitors in serum-free methylcellulose clonal cultures. Serial observations of the cultures showed hastening of growth of colonies by G-CSF, as determined by evaluating the time for individual colonies of 20 cells to reach 40 cells. G-CSF did not affect the incidence of proliferating cells in each developing colony. Cell-cycle analysis revealed that addition of G-CSF to cultures led to a decrease in the percentage of cells in the G1 phase of the cell-cycle, thereby indicating that G-CSF can modulate the cell-cycle of hematopoietic progenitors mainly by shortening the period of the G1 phase. Tumor necrosis factor alpha (TNF alpha) exerted opposite effects on cell-cycling of hematopoietic progenitors to those seen with G-CSF. G-CSF abolished the inhibitory effects of TNF alpha on the cell-cycling of hematopoietic progenitors. These observations indicate positive and negative regulatory roles of C-CSF and TNF alpha, respectively, and their interactions in the regulation of cell-cycling of hematopoietic progenitors.

Using polymerase chain reaction and single strand conformation polymorphism assay, we examined genetic alterations of p53 and K-ras genes in multiple lesions of colorectal cancers that developed in three patients with ulcerative colitis. p53 gene mutations were found in two lesions and a K-ms gene mutation at codon 12 was found only in one. Neither was found in non-cancerous lesions. The results indicate that mutation of both genes may not be so frequent among Japanese patients. They were found only in very few cancerous lesions of each patient. Therefore, it is possible that the oncogenic process in each lesion is a consequence of rather independent events. Our preliminary results suggest the potential usefulness of CD44 variant forms as markers for dysplastic and cancerous tissues in ulcerative colitis.