Didox, a ribonucleotide reductase inhibitor, induces apoptosis and inhibits DNA repair in multiple myeloma cells.

Ribonucleotide reductase (RR) is the enzyme that catalyses the rate-limiting step in DNA synthesis, the production of deoxynucleotides. RR activity is markedly elevated in tumour tissue and is crucial for cell division. It is therefore an excellent target for cancer chemotherapy. This study examined the anti-myeloma activity of Didox (3,4-Dihydroxybenzohydroxamic acid), a novel RR inhibitor (RRI). Our data showed that Didox induced caspase-dependent multiple myeloma (MM) cell apoptosis. Didox, unlike other RRIs that mainly target the pyrimidine metabolism pathway, targets both purine and pyrimidine metabolism pathways in MM, as demonstrated by transcriptional profiling using the Affymetrix U133A 2.0 gene chip. Specifically, a >or=2-fold downregulation of genes in these anabolic pathways was shown as early as 12 h after exposure to Didox. Furthermore, apoptosis was accompanied by downregulation of bcl family proteins including bcl-2, bcl(xl), and XIAP. Importantly, RR M1 component transcript was also downregulated, associated with decreased protein expression. Genes involved in DNA repair mechanisms, specifically RAD 51 homologue, were also downregulated. As Didox acts on MM cells by inhibiting DNA synthesis and repair, combination studies with melphalan, an agent commonly used in MM, were performed. A strong in vitro synergism was shown, with combination indices of <0.7 as determined by the Chou-Talalay method. These studies therefore provide the preclinical rationale for evaluation of Didox, alone and in combination with DNA-damaging agents, to improve patient outcome in MM.

Antimyeloma activity of two novel N-substituted and tetraflourinated thalidomide analogs.

Thalidomide alone or in combination with steroids has significant activity in multiple myeloma (MM). However, given its teratogenic potential, analogs have been synthesized, retaining the anti-MM activity without these side effects. We examined the anti-MM activity of two thalidomide analogs, CPS11 and CPS49. Direct cytotoxicity of the drugs on myeloma cell lines and patient myeloma cells was examined using thymidine uptake. Tumor cell apoptosis was evaluated by flow cytometry as well as Western blotting for caspase and PARP cleavage. Cellular signaling events were examined by immunoblotting for phosphorylated proteins. Both drugs inhibit proliferation of several MM cell lines sensitive and resistant to conventional therapies. They decrease secretion of IL-6, IGF, and VEGF by marrow stromal cells. Importantly, they inhibit proliferation of MM cells adherent to stromal cells. These drugs induce caspase-mediated apoptosis in MM cell lines, as well as patient MM cells. They inhibit the PI3K/Akt and JAK/STAT (signal transducers and activators of transcription) pathways in MM cells and are antiangiogenic in matrigel-based assays. CPS11 and CPS49 have potent antimyeloma activity and can overcome protective effects of the tumor microenvironment. They have potent antiangiogenic activity and direct effect on bone marrow stroma. These encouraging preclinical data provide the basis for further evaluation in the clinic.

Arsenic trioxide and the growth of human T-cell leukemia virus type I infected T-cell lines.

A novel therapeutic potential for acute promyelocytic leukemia using arsenic trioxide (As(2) O(3) ) has been reported. Recent in vitro studies demonstrated that As(2) O(3) effectively inhibits the growth of some cell lines derived from patients with malignant lymphoma, chronic lymphocytic leukemia and multiple myeloma. Adult T-cell leukemia (ATL) is an aggressive neoplasm of mature T-cell origin caused by human T-cell leukemia virus type-I (HTLV-I) the prognosis of which still remains very poor. A possible role of As(2) O(3) for the treatment of ATL is demonstrated from evidence that As(2) O(3) significantly inhibits the growth of HTLV-I infected T-cell lines and induces apoptosis in fresh ATL cells at clinically achievable concentration of the agent. The growth inhibition of As(2) O(3) treated HTLV-I infected T-cell lines was induced by both apoptosis and G(1) phase accumulation. Cleaved bcl-2 protein and an enhanced expression of bak protein in the cells were coincidentally observed during As(2) O(3) treatment. A broad spectrum caspase inhibitor, z-Val-Ala-DL-Asp-fluoromethylketone inhibited the apoptosis induced by As(2) O(3). Increased expression of p53, Cip1/p21 and Kip1/p27, and dephosphorylation of retinoblastoma protein (pRb) were detected in the As(2) O(3) treated cells. In conclusion, As(2) O(3) might become a new therapeutic tool in the treatment of ATL as As(2) O(3) induces apoptosis by destruction of the bcl-2 protein and enhancement of the bak protein production proceeding to activate caspases, and also induces G(1) phase accumulation by enhancement of p53, Cip1/p21, Kip1/p27 and dephosphorylation of pRb to HTLV-I infected T-cell lines.

Arsenic trioxide inhibits growth of human T-cell leukaemia virus type I infected T-cell lines more effectively than retinoic acids.

Adult T-cell leukaemia (ATL) is difficult to cure using conventional therapies. Recently the therapeutic possibility of retinoic acids (RA) has been reported. In this study, suppression of in vitro growth of human T-cell leukaemia virus type I (HTLV-I) infected T-cell lines and fresh ATL cells by arsenic trioxide (As2O3) were evaluated by comparison with a series of RA derivatives. Proliferation of four HTLV-I-infected T-cell lines was significantly reduced within 72 h by 1.0 micromol/l As2O3. Growth of two out of four HTLV-I-infected T-cell lines was also inhibited by 1.0 micromol/l RA, but to a lesser extent than by As2O3. The mechanism of this growth inhibition was due to the induction of apoptosis. Apoptosis was also induced in fresh ATL cells from patients by AS2O3, but far less by RA. As described in patients with acute promyelocytic leukaemia, 1.0 micromol/l of As2O3 can be safely achieved in the serum of patients; however, it is difficult to maintain this concentration of RA. In conclusion, As2O3 has therapeutic potential for the treatment of ATL and may be far more clinically beneficial than RA.

Prevention of hypoxia-induced apoptosis by the angiogenic factor thymidine phosphorylase.

The angiogenic factor platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is expressed at higher levels in a wide variety of solid tumors compared to adjacent normal tissues. Patients with PD-ECGF/TP-positive colon and esophageal tumors have a poorer prognosis than those with negative tumors. The expression of PD-ECGF/TP is a prognostic factor independent of microvessel density suggesting that TP has effects on tumor progression independent of its angiogenic activity. Evidence that hypoxia and apoptosis affect tumor growth prompted us to determine whether increased expression of PD-ECGF/TP prevents apoptosis induced by hypoxia. KB/TP cells transfected with a PD-ECGF/TP cDNA were resistant to hypoxia-induced apoptosis. Among the degradation products of thymidine produced by PD-ECGF/TP, 2-deoxy-D-ribose and thymine partially prevented hypoxia-induced apoptosis. The ability of 1 microM 2-deoxy-D-ribose in combination with the same concentration of thymine to prevent hypoxia-induced apoptosis was similar to that of the overexpressed TP in KB cells. A concentration of 1 microM 2-deoxy-L-ribose abrogated the effects of these degradation products of thymidine. These findings suggested that TP can confer resistance to apoptosis induced by hypoxia and the degradation products of thymidine are involved in this resistance. Expression of PD-ECGF/TP may play an important role in the progression of solid tumors, and inhibitors of TP and analogs of the degradation products of thymidine may suppress the growth of tumors by promoting apoptosis.

[Adult T-cell leukemia complicated with intestinal tuberculosis].

We report a patient with adult T-cell leukemia (ATL) complicated with intestinal tuberculosis. A 57-year old man was admitted to our hospital because of fever and dyspnea. He was diagnosed as ATL by leukocytosis [leukocyte count 18,200/microliters with 56% of abnormal lymphocytes which express CD4(+) and CD25(+)] and seropositive result of anti-HTLV-1 antibody. Combination chemotherapy for ATL improved his serum LDH level and peripheral lymph nodes, but fever was still persistent. He had an emergency operation because of perforation of the cecum during the chemotherapy. Histological examination of the resected cecum revealed caseous necrosis and numerous mycobacterium, which induced a diagnosis of intestinal tuberculosis. Although there have been several reports on pulmonary tuberculosis in patients with ATL, this is the first report of intestinal tuberculosis in ATL as far as we know. We conclude that if the patients with ATL have persistent fever of unknown origin, we should take account of intestinal tuberculosis as one of differential diagnosis.

Significance of elevated levels of soluble factors in the cerebrospinal fluid in patients with adult T-cell leukemia.

Although it has been recognized previously that several markers are present in the cerebrospinal fluid (CSF), their clinical usefulness of these markers in the diagnosis of malignant lymphoma infiltrating to the CNS has not yet been established. In order to determine their diagnostic usefulness as markers of meningeal infiltration by lymphoma cells in patients with adult T-cell leukemia (ATL), we measured some soluble factors in the CSF of patients with ATL and non-ATL patients. Soluble CD4 (sCD4) was highly elevated in all patients with ATL and meningeal infiltration. The CSF level of the soluble interleukin-2 receptor (sIL-2R; sCD25) was markedly elevated in 13 (72.2%) of 18 patients with ATL and meningeal infiltration. Levels of sCD4 and sCD25 in the CSF of patients with ATL and meningeal infiltration were significantly higher than in non-ATL patients (p < .01 and p < .001, respectively). These findings indicate that levels of sCD4 and sCD25 in the CSF are probably associated with meningeal infiltration by leukemia cells expressing CD4 and CD25 on surface membranes. CSF levels of sCD4 in 14 (60.9%) of 23 ATL patients and sCD25 in 13 (72.2%) of 18 ATL patients without meningeal infiltration were moderately elevated. These findings suggest that a small number of leukemic cells which were not detected by conventional CSF examination may have infiltrated the meninges in these patients. Sequential measurements of sCD4 and sCD25 in CSF obtained from patients with meningeal infiltration by leukemic cells showed that sCD4 and sCD25 levels reflected the activity of leukemic meningitis and correlated with the number of cells in CSF. However, the levels of sCD4 in CSF did not fall below the limit of detection even when the number of cells in CSF became normal. It is thought that the level of sCD4 in CSF is a more sensitive marker for detecting the infiltration of leukemic cells in CSF than the number of cells present in the CSF considering the clinical course of two patients with acute type ATL. Therefore, ATL patients with meningeal infiltration should receive treatments until sCD4 levels become normal and not just until the number of cells become normal. Our results also suggest that measurement of CSF levels of sCD4 and sCD25 is useful for the differential diagnosis of aseptic meningitis and meningeal infiltration by leukemic cells in patients with smoldering ATL. We conclude that measurement of soluble factors in CSF plays an important role in diagnosis, prophylaxis and treatment of meningitis in patients with ATL.

Expression of the intestinal T-lymphocyte-associated-molecule recognized by the HML-1 antibody on mononuclear cells from HTLV-I-infected subjects.

We investigated the expression of a monoclonal antibody (HML-1) defined antigen that appears on human intestinal T-lymphocytes in HTLV-I-related disease. We studied 25 ATL, and 24 healthy HTLV-I carriers. Patients with acute ATL showed a variety of the expression of the HML-1 antigen (range 0.4-74.8%). HML-1 expression on mononuclear cells (MNCs) in blood from patients with chronic ATL ranged from 1.7-43.6% (mean 13.5%). This level of expression was less than that of patients with acute ATL, but not significantly. In patients with smoldering ATL, the degree of patients with acute ATL, but not significantly. In patients with smoldering ATL, the degree of expression ranged from 1.6-13.3% (mean 8.0%). In contrast to patients with acute ATL, MNCs from patients with acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), and B-cell type chronic lymphocytic leukemia (B-CLL) did not express the HML-1 antigen, except for the 2 patients with ALL. Healthy HTLV-I carriers and healthy controls also were negative for HML-1 reactivity. In acute ATL, patients with gastrointestinal tract infiltration tended to have high expression of the HML-1 epitope. After stimulation with phytohemagglutinin (PHA), healthy HTLV-I carriers showed significantly increased expression of the HML-1 epitope (P < 0.05). Recently, the beta 7 integrin family has been found to play a specific role in mucosal localization or adhesion, and HML-1 protein was found to match the deduced beta 7 N-terminal sequence. We propose that the cellular gene responsible for HML-1 epitope expression may, like IL-2, IL-2R, etc., be transactivated by infection with HTLV-I, and HML-1 antigen gene expression by HTLV-I infection may lead to infiltration of ATL cells with highly expressed HML-1 epitope into the gut mucosa.

Combination chemotherapy (RCM protocol: response-oriented cyclic multidrug protocol) for the acute or lymphoma type adult T-cell leukemia.

43 patients with the acute or lymphoma type ATL were treated with the new combination chemotherapy (RCM protocol: response-oriented cyclic multidrug protocol) between January 1989 and December 1991. Complete response (CR) and partial response (PR) were achieved in 20.9% and 65.1% of all treated patients respectively. The median duration of survival was 6.0 months. The survival duration of patients with a high serum lactate dehydrogenase (LDH) value (> or = 1,000 unit) and/or a poor performance status (PS) (PS 3 or 4) were also improved but not in patients with a severe leukocytosis (> or = 35,000/microliters). Toxicity was mild (grade 1 or 2) except hematologic toxicity in 4 patients (9.3%) and alopecia in one patient (2.3%). In spite of many patients with a poor PS (PS 3 or 4), our chemotherapeutic results are equal or superior to other previous reports. It seems that response-oriented chemotherapy is suitable for the ATL patients with poor prognostic factors. These results indicate that the RCM protocol is very useful as the first choice chemotherapy for the acute or lymphoma type ATL.

Elevated soluble CD4 levels in the cerebrospinal fluid in patients with adult T-cell leukemia.

Levels of the soluble form of the leukocyte surface antigen CD4 (sCD4) were measured by enzyme linked immunosorbent assay (ELISA) in the cerebrospinal fluid (CSF) of patients with adult T-cell leukemia (ATL) and other malignant and non-malignant diseases. All patients with ATL and meningeal infiltration had markedly elevated levels of sCD4 in the CSF (53.7 +/- 34.9 U/ml). ATL patients without CSF pleocytosis often had elevated levels of sCD4 (15.1 +/- 9.2 U/ml). Non-ATL patients with CSF pleocytosis had elevated levels of sCD4 (23.3 +/- 12.2 U/ml) and those without CSF pleocytosis also showed elevation of sCD4 levels (16.8 +/- 9.3 U/ml). However, the mean levels of sCD4 in CSF from these patients were significantly lower than ATL patients with meningeal infiltration. Soluble CD4 in the CSF from healthy volunteers were below the detectable limit. We conclude that meningeal infiltration of CD4(+) ATL cells is strongly associated with elevated sCD4 levels in CSF, and some part of sCD4 in CSF may be originated from the native cells in the CNS as a response of inflammatory stimulations. Therefore, measurement of sCD4 may be useful in the diagnosis of meningeal infiltration and/or meningeal irritation in patients with ATL.

[Oral administration of low-dose etoposide for maintenance chemotherapy in adult T cell leukemia patients].

We studied the effectiveness of low-dose, oral administration of etoposide for maintenance chemotherapy of patients with adult T cell leukemia (ATL). Sixteen patients (9 males and 7 females) in remission (9 in complete remission and 7 partial remission) were orally administered 25 or 50 mg/day of etoposide. Median response duration of all the patients to the therapy was 18.6 months, ranging from 5.0 to 34.0 months. Thirteen out of the 16 patients relapsed, and 9 of them died of tumor progression. Appetite loss occurred in one case, without any other severe side effects. It has been suggested, therefore, that oral administration of the etoposide is useful for maintenance chemotherapy in ATL patients.

Tumor necrosis factor-beta in the serum of adult T-cell leukemia with hypercalcemia.

Serum tumor necrosis factor-beta (TNF-beta) from patients with adult T-cell leukemia (ATL) was studied by a sandwich enzyme-linked immunosorbent assay (ELISA) developed in our laboratory using biotinylated monoclonal anti-TNF-beta and recombinant TNF-beta. Seven of eight patients with hypercalcemia showed elevation of serum TNF-beta. On the other hand, TNF-beta could not be detected by the ELISA in 28 patients without hypercalcemia. The lower detection limit in this assay was 100 pg/mL, corresponding to 500 pg/mL by the conventional method. In two patients serum TNF-beta level decreased after treatment in association with the level of serum calcium. Furthermore, immuno-staining using anti-TNF-beta and avidin-biotin complex showed the presence of cytoplasmic TNF-beta in not only human T-cell leukemia virus type I infected cell lines, but also freshly isolated cells from ATL patients with hypercalcemia. The actual biologic activity of TNF-beta in serum was confirmed by a conventional bioassay in a patient with hypercalcemia, and its cytotoxic activity was inhibited by the addition of anti-TNF-beta antibody in the assay. These results suggested that serum TNF-beta might be one of the factors contributing to the hypercalcemia, at least in patients with ATL.

In vivo and in vitro effects of zinc oxide-eugenol (ZOE) on biosynthesis of cyclo-oxygenase products in rat dental pulp.

To determine the in vivo effects of a zinc oxide-eugenol mixture (ZOE) on the cyclo-oxygenase system in dental pulp, we used radioimmunoassay to measure the levels of prostaglandin E2 (PGE2), 13,14-dihydro-15-keto-PG (DHK-PG), thromboxane B2 (TXB2), and 6-keto-PGF1 alpha in the dental pulp of rats. When the dental pulp was irritated by a hole made in the dentin of the mandibular incisors without use of any coolants, the levels of these cyclo-oxygenase products in the pulp were increased to, respectively, 2.8, 1.7, 10.0, and 2.6 times those in the normal pulp at six hr after treatment. In contrast, these increases in cyclo-oxygenase products disappeared immediately when the artificial cavity in the dentin was filled with ZOE (P/L; 1 g/0.25 mL), but were not altered when the cavity was filled with zinc oxidewater (ZOW, 1 g/1.5 mL). Most of the eugenol portion of ZOE was released into the pulp within two hr after the cavity was filled with ZOE. The maximal eugenol content was 35 pmol per mg of pulp. Furthermore, when the cavity was filled either with ZOE or by the addition of 10 mumol/L eugenol to the pulp homogenate, biosynthesis of 14C-6-keto-PGF1 alpha, PGF2 alpha, and PGE2 from 14C-arachidonic acid in the homogenate was inhibited. These results suggest that eugenol released from ZOE in the cavity prepared in the dentin inhibited the biosynthesis of cyclo-oxygenase products during pulp irritation.