Antitumor effect of beta2-microglobulin in leukemic cell-bearing mice via apoptosis-inducing activity: activation of caspase-3 and nuclear factor-kappaB.

We have reported previously that beta2-microglobulin (beta2m) induces apoptosis in leukemic cells in vitro, and that an interaction between beta2m and HLA class I antigen induces apoptosis. Here we examined whether beta2m can induce apoptosis in leukemic cells in vivo and whether it has an antitumor effect in tumor-bearing mice. Daily administration of 50 or 250 microg of beta2m induced apoptosis and an antitumor effect on K562 leukemia cell-bearing mice in the same manner as tumor necrosis factor-alpha. In tumor tissues in beta2m-treated mice, both caspase-3 and nuclear factor-kappaB (NF-kappaB) were stained more strongly than in control mice by anti-caspase-3 and anti-NF-kappaB p65/Rel A polyclonal antibodies. We also observed the in vivo immunological effects of beta2m on lymphoid and hematopoietic organs, such as thymus, bone marrow, Peyer's patches, liver, and spleen in normal mice. Using antibodies against caspase-3 and NF-kappaB, immunohistochemical staining showed that no specific tissues were damaged or stained in normal mice. We conclude that beta2m stimulates caspase-3 and NF-kappaB pathways to induce apoptosis, making it a useful approach to a new therapy for leukemia.

A new apoptotic pathway for the complement factor B-derived fragment Bb.

Apoptosis is involved in both the cellular and humoral immune system destroying tumors. An apoptosis-inducing factor from HL-60 myeloid leukemia cells was obtained, purified, and sequenced. The protein found has been identified as a human complement factor B-derived fragment Bb, although it is known that factor B is able to induce apoptosis in several leukemia cell lines. Monoclonal antibodies against fragment Ba and Bb inhibited the apoptotic activity of factor B. When the purified fragment Bb was used for apoptosis induction, only the anti-Bb antibody inhibited Bb-induced apoptosis, and not the anti-Ba antibody. The apoptosis-inducing activity was found to be enhanced under conditions facilitating the formation of Bb. Blocking TNF/TNFR or FasL/Fas interactions did not interfere with the factor B-induced apoptosis. CD11c (iC3bR) acts as the main subunit of a heterodimer binding to fragment Bb in the apoptosis pathway, and the factor B-derived fragment Bb was found to possess the previously unknown function of inducing apoptosis in leukemic cells through a suicide mechanism of myeloid lineage cells during the differentiation stage.

NH2-terminal pentapeptide of endothelial interleukin 8 is responsible for the induction of apoptosis in leukemic cells and has an antitumor effect in vivo.

We have reported that endothelial interleukin 8 (IL-8) induces apoptosis in leukemic cells in vitro and in vivo, and that interaction between endothelial cells and leukemic cells causes induction of apoptosis through the release of endothelial IL-8 (Y. Terui et al., Biochem. Biophys. Res. Commun., 243: 407-411, 1998; Y. Terui et al., Blood, 92: 2672-2680, 1998). Here, we examined whether a pentapeptide corresponding to the NH2-terminal region of endothelial IL-8 can induce apoptosis in leukemic cells. The NH2-terminal pentapeptide Ala-Val-Leu-Pro-Arg (AVLPR) was found to significantly induce apoptosis in the leukemic cell lines K562, HL-60, Jurkat, and Daudi, as compared with the COOH-terminal pentapeptide Arg-Glu-Ala-Asn-Ser (REANS). Moreover, the NH2-terminal pentapeptide AVLPR significantly inhibited growth of i.p. and s.c. tumor masses of K562 cells and induced apoptosis in these cells in vivo. The active site of endothelial IL-8 is the NH2-terminal pentapeptide AVLPR, and this may serve as a new therapy for hematological malignancies.

[Moderate aplastic anemia associated with Crohn's disease during antithymocyte globulin treatment].

A 53-year-old woman with moderate aplastic anemia (AA) was treated with antithymocyte globulin (ATG). However, on the 4th day of treatment, ATG was discontinued because of bloody vomiting and melena. The patient improved with conservative treatment but complained of abdominal pain when the prednisolone (PSL) dose was decreased. Crohn's disease was finally diagnosed on the basis of upper and lower gastrointestinal X-ray studies. The patient responded well to ATG with hematologic improvement, and maintained remission with low-dose PSL and nutritional support. Drug-induced AA may occur during treatment for Crohn's diseases. The association of AA and Crohn's disease is rare, and to our knowledge, has not yet been reported in the literature. We discussed the pathogenesis of Crohn's disease during immunotherapy for AA.

Beta(2)-microglobulin identified as an apoptosis-inducing factor and its characterization.

Major histocompatibility complex (MHC) molecules play an important role in antigen presentation for induction of tumor as well as cellular and humoral immunities. Recent studies using anti-MHC antibodies demonstrated that antibodies specific for HLA class I molecules induced cellular activation and a type of apoptosis that may be distinct from Fas-dependent or TNFR (tumor necrosis factor-alpha receptor)-dependent processes. We purified a previously untested apoptosis-inducing factor from HL-60 human leukemic cell-conditioned media to homogeneity and sequenced it. It was identified as beta(2)-microglobulin (beta(2)m), which has been previously known as thymotaxin and is a part of the HLA class I antigen complex. beta(2)m acts on both T-leukemic cells and myeloid leukemic cells to induce apoptosis, which then activates caspase 1 and 3. Cross-linking studies showed that biotinilated beta(2)m recognized an epitope distinct from those recognized by the anti-HLA class I antibody, as reported previously. We demonstrated that beta(2)m plays a previously unrecognized and important role in regulating the elimination of tumor cells, which occurs as a result of the action of beta(2)m as an apoptosis-inducing factor.

Activated endothelial cells induce apoptosis in leukemic cells by endothelial interleukin-8.

Tumor cells are eradicated by several systems, including Fas ligand-Fas and tumor necrosis factor (TNF)-tumor necrosis factor receptor (TNFR). In the previous study, we purified an apoptosis-inducing factor (AIF) to homogeneity from a medium conditioned by PDBu-treated HL-60 cells. N-terminal sequence analysis showed that AIF is identical to endothelial interleukin-8 (IL-8). A novel apoptosis system, in which endothelial cells participate via endothelial IL-8 release, is identified here. Human umbilical vein cells (VE cells) produce and secrete IL-8 by stimulation of IL-1alpha and TNF-alpha. Endothelial IL-8, which is secreted from VE cells by stimulation of IL-1alpha and TNF-alpha , induces apoptosis in myelogenous leukemia cell line K562 cells. Monocyte-derived IL-8 could not induce apoptosis in K562 cells. Moreover, interaction between VE cells and K562 cells induces the release of endothelial IL-8 from VE cells, and the attached K562 cells undergo apoptosis. Moreover, interactions between VE cell and other cell lines, such as HL-60, U937, Jurkat, and Daudi, induce the secretion of endothelial IL-8 and the induction of apoptosis in cell lines. Endothelial IL-8 significantly inhibits tumor growth of intraperitoneal and subcutaneous tumor mass of K562 cells and induces apoptosis in their cells in vivo. Endothelial IL-8 plays an important role in apoptosis involving endothelial cells, which may provide us with a new therapy for hematological malignancies.

Identification of a novel apoptosis-inducing factor derived from leukemic cells: endothelial interleukin-8, but not monocyte-derived, induces apoptosis in leukemic cells.

The human myelogenous leukemia cell line HL-60, treated with phorbol 12, 13-dibutyrate (PDBu), produces apoptosis-inducing factors (AIFs) in leukemic cells. We have purified AIF against leukemic cell line K562 as target cells, and N-terminal amino acid sequencing analysis revealed that this purified protein is identical to endothelial cell-derived interleukin-8 ([(Ala)-IL-8]77). In Western blot analysis of supernatants of PDBu-treated HL-60 cells, only [(Ala)-IL-8]77 was detected. Moreover, recombinant human [(Ala)-IL-8]77 induced apoptosis in leukemic cell lines such as K562, HL-60, KG-1, U937, THP-1 and Jurkat, but monocyte-derived IL-8 ([(Ser)-IL-8]72) did not. Therefore [(Ala)-IL-8]77 plays an important role in inducing apoptosis against leukemic cells and may lead to a new therapy for leukemia.

Rare but important adverse effects of all-trans retinoic acid in acute promyelocytic leukemia and their management.

Several adverse effects have been reported to occur after clinical application of all-trans retinoic acid (RA) in acute promyelocytic leukemia (APL). Except for severe side effects including retinoic acid syndrome, the mechanism of action of RA on adverse effects remains unclear. Here we describe some rare adverse effects and their management. We reviewed the English literature, and we added our cases of endocrine and metabolic adverse effects, such as hypercalcemia, male infertility, bone marrow necrosis, fibrosis and acute pancreatitis. We also described our cases of thromboembolic events, RA-dependent growth of pathologic cells including Sweet's syndrome, erythema nodosum, hyperhistaminemia, granulomatous proliferation, and mild cases of pulmonary complications. In addition, we reviewed the efficacy of RA administration for other types of leukemia or myelodysplastic syndrome. RA and chemotherapeutic agents might induce complete remission, but we obtained a response in only one case of M2 in the third relapse. During RA administration the patient should be monitored for these adverse effects, and early diagnosis and appropriate treatment are important.

Expression of Src-like adapter protein mRNA is induced by all-trans retinoic acid.

By using a differential display method, specific bands were selected from ladder PCR products derived from ATRA-dependent differentiated U937 cells, in comparison with those of untreated U937. By screening the cDNA library of ATRA-dependent differentiated U937 cells with one of the PCR products, we cloned the src-like adapter protein (SLAP). Northern blot analysis of U937 cells with or without ATRA treatment indicated that the SLAP mRNA was clearly induced by ATRA. The induction was inhibited by the addition of cycloheximide, indicating that ATRA acted indirectly through synthesis of other proteins. The SLAP mRNA was induced in HL60 and NB-4 but not in K562 or THP-1. Interestingly, these cells in which SLAP mRNA was induced by ATRA all showed ATRA-dependent cell differentiation. The relationship between SLAP and cell differentiation is unclear, but SLAP may transduce a signal for cell differentiation.

Tretinoin induces bone marrow collagenous fibrosis in acute promyelocytic leukaemia: new adverse, but reversible effect.

In 11/13 acute promyelocytic leukaemia (APL) cases treated with tretinoin (RA) we observed RA-induced inaspirable collagenous fibrosis of the bone marrow. To study the mechanism of RA on collagen production, we cultured a human bone marrow derived stromal cell line and an osteoblastic cell line with RA in vitro. 10(-7) and 10(-6)M of RA stimulated collagen production. Clinical and experimental observation may be important to understand this adverse effect of RA as it is useful in cancer chemoprevention as well as treatment for APL. This adverse effect is spontaneously reversible after stopping RA or following chemotherapy.

Monocyte chemoattractant protein-1 stimulates tumor necrosis and recruitment of macrophages into tumors in tumor-bearing nude mice: increased granulocyte and macrophage progenitors in murine bone marrow.

Monocyte chemoattractant protein-1 (MCP-1) belongs to the newly recognized "chemokine" superfamily of activation-inducible cytokines. We report here that MCP-1 gene-transferred mouse myeloma cells modulate tumor necrosis in myeloma-bearing nude mice. We established an MCP-1-producing myeloma cell line (X63-MCP-1) by transfection with human MCP-1 cDNA as well as interleukin-8-producing X63 cells (X63 IL-8). Each cell line showed the same growth characteristics in vitro, and 1 x 10(7) cells per mouse were injected into the peritoneal cavity resulting in the formation of tumors. Hematologic studies, including peripheral white blood cell counts and differentiation, showed no differences among the groups. They formed tumors in the same manner, which we observed from weeks 2.5 to 9. MCP-1 mice showed more tumor necrosis and infiltration of the macrophages into the tissue surrounding the tumor. In situ hybridization, using a partial cDNA as a probe, showed that macrophages contained MCP-1 mRNA. Bone marrow cell colony-forming assay showed a greater number of both granulocyte and macrophage colonies in MCP-1 mouse femur than in those of controls or interleukin-8 mice. MCP-1 has no direct stimulatory activity on stem cells, but longer exposure to MCP-1 in vivo might stimulate both granulocyte and macrophage progenitors and recruitment of macrophages into tumors, and it might explain the antitumor activity of macrophages in tumor-bearing nude mice.