Short exposure to actinomycin D induces "reversible" translocation of protein B23 as well as "reversible" inhibition of cell growth and RNA synthesis in HeLa cells.

HeLa cells were grown in medium containing various amounts of actinomycin D for various times. Cellular localization of protein B23 was detected using an immunofluorescence technique. Bright nucleolar fluorescence was observed in untreated cells. A shifting of nucleolar to nuclear fluorescence was observed with increasing doses of actinomycin D and longer incubation times. The degree of translocation of protein B23 from nucleoli to nucleoplasm is dependent on the amount of the drug used and the duration of incubation. Short exposure (0.5 h) of HeLa cells to actinomycin D (0.01-0.25 microgram/ml) induced "reversible" translocation of protein B23, inhibition of cell growth, and RNA synthesis. A majority of cells (greater than 75%) treated with actinomycin D (0.01-0.25 microgram/ml) for 0.5 h still retained bright nucleolar fluorescence. A shifting of nucleolar to nuclear fluorescence as well as inhibition of cell growth and RNA synthesis were observed within 6 h after the removal of the drug. However, at the extended periods (greater than 24 h) after drug removal, RNA synthesis and cell growth resumed at the normal rate, and protein B23 relocated from nucleoplasm to nucleoli. This is in contrast to the results obtained from the experiments using higher doses (1 microgram/ml; 0.5 h) or longer (0.25 microgram/ml; 2 h) exposure of HeLa cells to actinomycin D, which induced irreversible B23 translocation as well as irreversible inhibition of cell growth and RNA synthesis. These results indicated that actinomycin D can be a reversible inhibitor depending on the drug extracellular concentrations and exposure times. Our results also indicated that "B23 translocation" is closely associated with states of cell growth and inhibition of RNA synthesis. "B23 translocation" may therefore be a simple and rapid method for assessing the inhibition of cell growth in response to antitumor therapy.     

Inhibition of RNA and protein syntheses makes non-differentiating mouse myeloid leukemia cells sensitive to a factor(s) stimulating differentiation.

Treatment with ascitic fluid from animals bearing various tumors, can induce mouse myeloid leukemia line cells, M1, to differentiate in vitro into macrophages and granulocytes. Cells were isolated that were resistant to the ascitic fluid factor(s) stimulating differentiation (D-factor). The resistant cells became sensitive to the D-factor and differentiated after treatment with various inhibitors of RNA synthesis (actinomycin D, nogalamycin, chromomycin A3 or cordycepin) or protein synthesis (puromycin or cycloheximide). The cells could not be induced to differentiate by treatment with the inhibitors alone. The effective doses of the inhibitors of protein synthesis were toxic to the cells. Among these inhibitors actinomycin D (5 ng/ml) was the most effective for sensitizing the resistant cells. Inhibitors of DNA synthesis did not sensitize the resistant cells. Added actinomcyin D was mainly recovered in the nuclear fraction of the cells. The sensitizing effect of actinomycin D on the cells was roughly parallel to the extent of its inhibition of RNA synthesis in the cells. The effective concentration of actinomycin D (5 ng/ml) mainly inhibited it also inhibited alpha-amanitin-resistant RNA synthesis to some extent. These results suggest that alpha-amanitin-sensitive RNA synthesis may be involved in sensitization of the resistant cells to the D-factor.     

Induction of Apoptosis in MDA-231 Cells by Protein Synthesis Inhibitors Is Suppressed by Multiple Agents

In the present study we investigated the ability of several diverse agents to inhibit MDA-231 cell death induced by two different protein synthesis inhibitors, cycloheximide (CHX) and ricin. Cell death was evaluated by several techniques: trypan blue staining, determination of the released lactic dehydrogenase, transmission electron microscopy, and DNA fragmentation. Results from DNA gel electrophoresis and electron microscopy suggest a mechanism of death by apoptosis which terminates in necrosis. Approximately 60% of cell death was induced either by a continuous exposure to 30 μg/ml CHX for 48 hr or by a 1-hr exposure to 250pg/ml ricin followed by a subsequent incubation of 48 hr in the absence of the drug. Cell survival, in the protein synthesis-inhibited cells, was enhanced by the following diverse agents: the growth factors EGF (20 ng/ml) and IGF-1 (20 ng/ml), the protein kinase C activator 12-0-tetradecanoyl-phorbol-13-acetate (5 ng/ml), the protein kinase A activator 8-bromoadenosine 3':5'-cyclic monophosphate (650 μg/ml), the nuclease inhibitor aurintricarboxylic acid (100 μg/ml), and fetal bovine serum (5%). The survival agents that stimulated protein synthesis in the control untreated cells had no effect on the CHX—inhibited protein synthesis, which indicates that new protein synthesis is not required for cell survival. The same survival agents attenuated the continuous decrease in protein synthesis in the ricin-exposed cells; therefore, the involvement of new protein synthesis in the survival mechanism could not be excluded. The protein kinase C inhibitor staurosporine blocked, in a dose-dependent manner, the survival effect of 12-0-tetradecanoyl-phorbol-13-acetate and EGF, but not that of aurintricar-boxyclic acid or fetal bovine serum, in the protein synthesis-inhibited cells. These results provide evidence for several distinctive pathways, the activation of which inhibits MDA-231 cell death induced by protein synthesis inhibitors. Some of these pathways involved activation of protein kinases, probably protein kinase C.     

Effects of luzopeptins on protein B23 translocation and ribosomal RNA synthesis in HeLa cells

Localization of protein B23 in HeLa cells after treatment with luzopeptin A and its analogues was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with luzopeptin A (50 ng/ml), luzopeptin B (500 ng/ml), or luzopeptin D (10 ng/ml) for 2 h, uniform nucleoplasmic rather than specific nucleolar fluorescence was observed. Luzopeptin C had no effect on protein B23 translocation. Luzopeptin D, A, and B inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with 50% inhibitory concentration values of 3.7 +/- 1.1 (SD), 10.8 +/- 2.1, and 122.0 +/- 34.0 ng/ml, respectively. Less than 10% inhibition of [3H]uridine incorporation was found with luzopeptin C (500 ng/ml and 2 h incubation). Ribosomal RNAs (28 and 18S) were isolated from HeLa cells treated with luzopeptin D (50 ng/ml; 2 h). They were then separated and analyzed in 1% agarose gel electrophoresis. There were 90.1 +/- 1.38 and 95.0 +/- 1.04% inhibition of [3H]uridine incorporation into 28 and 18S ribosomal RNA, respectively. The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was luzopeptin D greater than luzopeptin A greater than luzopeptin B much greater than luzopeptin C, which correlates with the order of their 50% inhibitory concentration values for inhibition of [3H]uridine incorporation. With 34-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. With 70-85% inhibition of RNA synthesis, a uniform nucleoplasmic fluorescence was observed. These results indicate that translocation of protein B23 as observed by indirect immunofluorescence may be a rapid and simple screening test for the selection of antitumor agents which inhibit ribosomal RNA synthesis.     

All masquerading as aul

Of 597 cases of acute leukaemia in adults (>16 years) seen at St. Bartholomew's Hospital, London, between May 1973 and January 1982, 412 were diagnosed as AML, 103 as ALL and 58 as Philadelphia chromosome positive blast crisis of CML (13 presenting as acute leukaemia and 45 having a prior chronic phase). The remaining 24 cases were considered to be acute undifferentiated leukaemia. Twenty-one of the latter were investigated using a panel of immunological markers at diagnosis and/or retrospectively using frozen cell suspensions. Eighteen out of 21 were shown to have a predominantly ‘lymphoid’ phenotype which comprised 12 cases of common ALL (two of whom were Ph¹ positive), three cases of null-ALL, one case with a probable early thymic phenotype, and two cases with a monoclonal B lymphoblast phenotype. One ‘common ALL’ and one ‘null-ALL’ had a significant proportion of pre-B (cytoplasmic μ chain⁺) cells. One other case reacted with anti-myeloid sera. Leukaemic blasts from two patients were unreactive with all markers tested. No cases of glycophorin positive erythroleukaemia or anti-platelet (glycoprotein I) positive leukaemia were detected.

These observations suggest that the overwhelming majority of acute leukaemias have an identifiable affiliation to the lymphoid or myeloid lineages and that patients diagnosed haematologically as ‘AUL’ might benefit by therapy appropriate for their leukaemic cell type.     

Amphotericin B-induced sensitivity to actinomycin D in drug-resistant Hela cells.

HeLa cells, which were selected for resistance to actinomycin D on the basis of decreaded penetration of the antibiotic into the cells, were treated with nontoxic concentrations of the polyene antibiotic amphotericin B. In the presence of amphotericin B, the cells became sensitive to the effects of actinomycin D, as demonstrated by loss of cell viability, typical morphological changes, and effects on the pattern of RNA synthesis. Furthermore, we were able to demonstrate that amphotericin B increased the amount of (3H)actinomycin D incorporated into HeLa cells as determined by both radioactive counts and radioautography. Amphotericin B might be useful in overcoming the resistance is based on decreased entry of the agents into cells.     

Phosphoprotein B23 translocation and modulation of actinomycin D and doxorubicin cytotoxicity by dipyridamole in HeLa cells.

During continuous exposure, cells were more responsive to doxorubicin (DOX) in the presence of dipyridamole (DPM). Translocation of nucleolar phosphoprotein B23 and inhibition of cell growth occurred with a lower dose of DOX and in a shorter incubation time in the presence of DPM. DPM did not change translocation induced by actinomycin D (Act-D). Short exposure of HeLa cells to Act-D induced "reversible" translocation of protein B23 as well as "reversible" inhibition of cell growth. DPM included in the cell culture after removal of Act-D inhibited the recovery of cell growth as well as the corresponding relocalization of protein B23 from the nucleoplasm to nucleoli. DPM administered in the fresh medium after 30 min exposure to DOX had little effect on the potentiation of the induced translocation of protein B23 and inhibition of cell growth. Our results indicated that "B23 translocation" is closely associated with states of cell growth. The potentiation of the inhibition of cell growth by DPM is associated with the extent of enhanced protein B23 translocation. "B23 translocation" may therefore be a simple and rapid method for assessing the inhibition of cell growth and for determining the efficacy of combination cancer chemotherapy.     

Induction of differentiation of Rauscher virus-induced mouse myeloid leukemia cells with a factor(s) in ascitic fluid and inhibitors of nucleic acid and protein syntheses.

Cell line R453, established from a Rauscher virus-induced myeloid leukemia in a C57BL/6 mouse, was induced to differentiate in vitro into macrophages and granulocytes with ascitic fluids from animals bearing various ascites tumors or from mice treated with complete Freund's adjuvant, conditioned media from various cell lines, and glucocorticoid hormone. Differentiated R453 cells had a morphology similar to that of macrophages and granulocytes in normal hematopoietic organs, and they phagocytized small paricles such as latex particles, moved in soft agar showing locomotive activity, and had Fc and C3 receptors on the cell surface. This induction of differentiation of R453 cells was markedly enhanced by addition of inhibitors of RNA synthesis (actinomycin D, nogalamycin, or chromomycin A3), protein synthesis (puromycin or cycloheximide), or DNA synthesis (methotrexate, hydroxyurea, 5-fluorodeoxyuridine, or 1-beta-D-arabinofuranosylcytosine) in the presence of ascitic fluid. Of the inhibitors, actinomycin D was the most effective at a low concentration (5 ng/ml) in stimulating induction of differentiation of R453 cells. However, these inhibitors alone did not induce differentiation of R453 cells. The factor(s) in ascitic fluid that stimulates differentiation of R453 cells was heat labile, nondialyzable, and inactivated by trypsin.     

Induction of apoptosis in human leukemia cells by MCS-C2 via caspase-dependent Bid cleavage and cytochrome c release

Much of the interest on the chemopreventive properties of licorice has been focused on the plant genius Glycyrrhiza glabra. In this study the ethanol extract of Chinese licorice root, Glycyrrhiza uralensis (G. uralensis) was investigated for its estrogenic effect and the ability to inhibit cell proliferation in the MCF-7 human breast cancer cell line. The extract of the root of G. uralensis was fractionated in EtOH:H₂O (80:20) (80% ethanol). The extract exhibited estrogenic effects similar to 17β- estradiol (E2) and induced apoptosis at the same dose level (100 μg/ml) in MCF-7 breast cancer cells, results were associated with up-regulation of tumor suppressor gene p53 and pro-apoptotic protein Bax. G. uralensis extract caused the up-regulation of p21^waf1/cip1 and down-regulation of cdk 2 and cyclin E and most significantly, induced G1 cell cycle arrest. This is the first study to show that the ethanolic extract of the root of G. uralensis has an estrogen-like activity and anti-cancer effects against MCF-7 human breast cancer cells. Whilst the use of phytoestrogens to protect against hormone-dependent cancers or as a ‘natural’ alternative to hormone replacement therapy remains controversial, the data in this paper support the suggestion that extracts of root of the Chinese licorice G. uralensis might be of importance in this debate.     

Effects of a prospective antitumor agent, 1,4-bis(2''-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate, on cultured mammalian cells

The effects of 1,4-bis(2'-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate (CBH; NSC 57198) on cell viability, growth, progression through the cell cycle, survival, and differentiation were investigated in suspension cultures of murine lymphocytic leukemia (L1210) and erythroleukemic (FL) cells and normal human lymphocytes stimulated with phytohemagglutinin (PHA) and in adherent cultures of Chinese hamster ovary (CHO) cells. CBH was equally cytotoxic toward stationary and exponentially growing CHO cells. Cell viability was diminished by 50% following 24 hr exposure to approximately 50 μg CBH per ml. Treatment of quiescent human lymphocytes for 24 hr with up to 100 μg CBH per ml did not appreciably diminish cell viability though the subsequent stimulation of such lymphocytes with PHA was inhibited in a dose dependent fashion. L1210, FL cells, and PHA stimulated human lymphocytes were equally sensitive to CBH, 50% inhibition of growth was obtained following 24 hr treatment with 25 μg CBH per ml. Incubation for up to 48 hr with CBH did not result in differentiation of FL cells to mature hemoglobin containing cells. Constant exposure of L1210 cells and PHA-stimulated human lymphocytes to 10-50 μg CBH per ml resulted in accumulation of cells in G₂ + M phase; higher drug concentrations resulted in cell arrest in mid to late S phase and G₂ phase. A short 1-hr pulse of the drug resulted in a transient accumulation of L1210 cells in S and G₂ phases. However, cells recovered from a short pulse of drug and by 48 hr, both cell proliferation and the cell cycle distribution appeared normal. A detailed analysis of cell cycle progression of L1210 cells in the presence of the drug indicated that the duration of G₂ phase was extended at low concentrations (10 μg/ml) while the transit of cells through S was retarded with subsequent accumulation in late S and G₂ phase at higher (50 μg/ml) concentrations. Concomitant with cell arrest in S and G₂ phase an increase in cellular RNA content indicating unbalanced growth was observed. This state of unbalanced growth was reversible in cultures exposed to a 1-hr pulse of up to 100 μg CBH per ml; cellular RNA content returned to control values by 48 hr. No effect on nuclear chromatin as assayed by acid denaturation was observed. Though the exact mechanism of drug action is not known, the data are not incompatible with the drug acting as an alkylating agent.     

Inhibition of recovery from bleomycin-induced potentially lethal damage.

Mammalian cells are able to recover from bleomycin (Bleo)-induced potentially lethal damage. We examined the influences of inhibitors of DNA, RNA, or protein synthesis on that recovery. Cytosine arabinoside and cycloheximide did not inhibit recovery from Bleo-induced damage; however, actinomycin D (Act D) did inhibit it. At a dose that produced minimal cell killing and inhibited only RNA synthesis, inhibition of recovery by Act D was immediate and complete.     

Antiproliferative Activity of Mammalian Lignan Derivatives Against the Human Breast Carcinoma Cell Line, ZR-75-1

The effect of each of twelve mammalian lignun derivatives on the growth of human mammary tumor ZR-75-1 cells was examined. At a concentration less than 10 μ g/ml, tumor cell growth was inhibited from 18-68%. The effect of 2,3-dibenzylbutane-1,4-diol (hattalin) was found to be strongest, inhibiting growth by 50% at a concentration (EC₅₀ of 2.1 μ g/ml. Hattalin inhibited membrane Na^%, K^%-ATPase of canine kidney cortex. It also inhibited the ATPase of the plasma membrane fraction from both cultured cells and a section of human breast cancer tissue at a concentration ranging from 0.5 to 2.0 mM. However, only a few percent of membrane ATPase from either ZR-75-1 cells or breast carcinoma tissue was inhibited by 2.0 mM of ouabain, suggesting that the target ATPase of hattalin was other than ouabainsensitive ATPase. The relative incorporation of [³H]thymidine per 1 10⁵ cells into the acid-precipitable fraction of ZR-75-1 cells was not affected by 1-50 μg/ml of hattalin, while a marked decrease resulted from 1-10 μg/ml of 5-fluorouracil (5-FU). These results suggest that the suppressive effect of hattalin on tumor cell growth my not occur through inhibition of DNA synthesis but rather partly by inhibition of the plasma membrane ATPase other than Na^% and K^% -dependent ones.     

Prolymphocytic Leukemia: Malignancy of Activated B Cells with an Apoptosis-like Response to Anti-Human IgM Signal Transduction

B-prolymphocytic leukemia (PLL) resembles B-chronic lymphocytic leukemia (CLL) in the expression of surface IgM, but differs in morphology, cellular function, and clinical features, suggesting that PLL and CLL may be derived from distinct B cell subsets in B cell ontogeny. In studies of the signaling effect of anti-human IgM (anti-μ) on B malignancies, the divalent F(ab') anti-μ neither stimulated nor inhibited DNA synthesis of CLL cells but exerted a unique negative signaling effect and direct cytotoxicity on PLL cells in complement-free cultures with morphological cellular changes characteristic of a “programmed cell death” mechanism or apoptosis. The susceptibility of PLL cells to anti-μ-triggered cytotoxicity is similar to the negative signaling effect observed on “tolerogenic” normal B cells. The findings of activated B cell phenotypes in PLL, a high level of autonomous DNA synthesis activity in large “transformed” PLL cells, and mitogen-induced cellular transformation of CLL to PLL indicate that PLL may be a malignant transformation of metabolically activated “tolerogenic” B cells.     

Fibronectin and sialic acid levels in human meningiomas and gliomas

In this study, fibronectin and sialic acid levels have been assayed in human meningiomas and gliomas. The mean fibronectin and sialic acid levels for human meningiomas were 22.01 ± 9.70 μg/mg protein and 19.58 ± 4.89 μg/mg protein, respectively, and for human gliomas were 27.30 ± 13.70 μg/mg protein and 25.67 ± 11.60 μg/mg protein, respectively, versus 9.23 ± 5.40 μg/mg protein and 13.50 ± 4.30 μg/mg protein for normal brain tissues. Fibronectin and sialic acid levels were significantly higher in human meningiomas (P < 0.01 and P < 0.05) and gliomas (P < 0.001 and P < 0.01) than control group. Also the mean fibronectin and sialic acid levels were found to be 18.27 ± 7.08 μg/mg protein and 17.04 ± 6.25 μg/mg protein in Grade I–II and 32.60 ± 15.00 μg/mg protein ad 29.50 ± 11.60 μg/mg protein in Grade III–IV gliomas, respectively. Fibronectin and sialic acid levels were significantly higher in Grade III–IV gliomas than Grade I–II gliomas (P < 0.05).     

Tumor Cell Invasion of Basement Membrane in Vitro is Regulated by Amino Acids

Because most cancer deaths result from disseminated disease, understanding the regulation of tumor invasion and metastasis is a central theme in tumor cell biology. Interactions between extracellular matrices (ECM) and cellular microenvironment play a crucial role in this process. We have tested selected amino acids andpolyamines for their ability to regulate RL95-2 cell invasion through both intact human amniotic basement membrane and a novel human ECM (Amgel). Three major systems for neutral amino acid transport, systems L, A, and ASC, are operational in these neoplastic cells. Amino acids entering the cell via transport system A or N, i.e., (methyl amino)-isobutyrate (MeAIB) orAsn, markedly enhanced invasiveness of these human adenocarcinoma cells as measured by a standard 72-hr amnion or Amgel invasion assay. Addition of 2-amino-2-norborane carboxylic acid (BCH; 1 mM), a model substrate of the L transport system, caused a significant decrease in invasive activity when tested in the Amgel assay. Interestingly, Val lowers steady-state levels of MeAIB uptake and blocks the increase in cell invasion elicited by MeAIB. At the same time, these amino acids do not influence cell proliferation activity. Neither the charged amino acid Lys or Asp (not transported by A/N/L systems) nor the polyamines putrescine, spermidine, or spermine modulate invasiveness under similar experimental conditions. Moreover, the observed time-dependent stimulation of system A activity (cellular influx of MeAIB) by substrate depletion is prevented by the addition of actinomycin D (5 μM) or cycloheximide (100 μM), suggesting the involvement of de novo RNA and protein synthesis events in these processes. MeAIB treatment of tumor cells selectively increased the activities of key invasion-associated type IV collagen-ases/gelatinases. These results indicate that in the absence of defined regulators (growth factors or hormones), certain amino acids may contribute to the epigenetic control of human tumor cell invasion and, by extension, metastasis. We propose that amino acids, acting via specific signaling pathways, modulate phenotypic cell behavior by modulating the levels of key regulatory enzymatic proteins.     

Selective inhibition of rat liver nuclear RNA polymerase II by actinomycin D in vivo

It is well known that actinomycin D, a carcinogen, inhibits DNA-dependent RNA synthesis. The interpretation of this inhibition has been that this carcinogen binds specifically to the deoxyguanosine moiety of a DNA molecule, and thus blocks the template function. This paper presents evidence which suggests that this single mechanistic interpretation of actinomycin D action may not be adequate in eukaryotic cells. Thirty minutes after actinomycin D injection (250 microg/100 g body weight), rat liver nuclear RNA synthesis was inhibited by 81% and nucleolar RNA synthesis was inhibited by 98%. In order to determine whether this inhibition is due to an inhibition of DNA template function or of the RNA polymerase activity, the total nuclear free and engaged RNA polymerases were solubilized and the individual RNA polymerase species were partially purified by DEAE-Sephadex column chromatography. It was found that while the overall enzyme activities of RNA polymerase I and III were not affected, there was a selective inhibition of RNA polymerase II activity (42%). This result suggests that actinomycin D, like aflatoxin B1 and N-hydroxy-2-acetylaminofluorene, inhibits nuclear RNA synthesis at multiple sites; it inhibits nucleolar RNA synthesis by blocking the nucleolar DNA template function, and it inhibits messenger RNA synthesis by inhibiting at least partially the enzyme RNA polymerase II activity per se.     

Activation of MMP-2 by human GCT23 giant cell tumour cells induced by osteopontin,bone sialoprotein and GRGDSP peptides is RGD and cell shape change dependent

We show that osteopontin (OPN), bone sialoprotein (BSP) and GRGDSP peptides, in solution, induce activation of metalloproteinase-2 (MMP-2) secreted by human GCT23 giant cell tumour cells. Activation of MMP-2 is RGD sequence dependent, possibly involves anti-αVβ3 integrins, is preceded by a change from spread to rounded cell morphology and is mimicked by the actin depolymerising agent cytochalasin B. Cells that had spread on OPN, BSP and GRGDSP substrata failed to activate MMP-2, but subsequent addition of soluble GRGDSP induced rounding and MMP-2 activation. Activation induced by GRGDSP and cytochalasin B was cell mediated, inhibited by EDTA, tissue inhibitor of metalloproteinase-2 (TIMP-2) and carboxyl terminal MMP-2 consistent with a role for membrane type (MT)-MMP but did not involve urokinase, plasmin or thrombin activity. Activation induced by GRGDSP and cytochalasin B, but not cell rounding, was inhibited by herbimycin A, cycloheximide and actinomycin D, suggesting a role for tyrosine kinases, protein and RNA synthesis, but was not associated with changes in mRNA for MT-MMP-1, MMP-1, MMP-2, TIMP-1 or TIMP-2. GRGDSP and cytochalasin B enhanced levels of membrane-associated pro- and active form MMP-1 and MMP-2 but not MT-MMP-1, stimulated cell surface MMP-1 staining and induced that of MT-MMP-1, MMP-2 and TIMP-2. This was consistent with the possible relocation of constitutive MT-MMP-1 to the cell surface as a prerequisite for subsequent cell surface MMP-2/TIMP-2/MT-MMP-1 complex formation and to the potential induction of conditions favourable for reciprocal cell surface MMP-1/MMP-2 activation. Our data provide a novel insight into interactions between RGD containing bone matrices, GCT cells and MMPs of potential relevance to GCT pathology. Int. J. Cancer 77:82–93, 1998.© 1998 Wiley-Liss, Inc.     

Circulating immune complexes in chronic myeloid leukemia patients at various stages of the disease

Circulating immune complexes (CICs) in sera from patients suffering from chronic myeloid leukemia (CML) at initial diagnosis, in ‘remission’, at relapse and in blastic crisis have been quantitated using fluid [¹²⁵I]Clq binding assay in terms of per cent binding activity and μg/ml aggregated human globulin (AHG) equivalents. The Clq binding activity (Clq-BA) has been compared within the groups of CML patients in different phases of the disease as well as with sera obtained from normal healthy donors. The results showed that the mean Clq-BA was significantly increased in CML patients at initial diagnosis (25.74 ± 3.48, p < 0.001), in relapse (53.36 ± 6.9, p < 0.001) and in blastic crisis (60.5 ± 8.7, p < 0.001) when compared to control sera. Sera of ‘remission’ patients showed significant decrease in Clq-BA when compared to sera collected in active phases of the disease, however, the values were still significantly higher (12.87 ± 1.58, p < 0.02) than those of normal healthy donors. When the levels of CICs as assessed by Clq-BA were compared with the WBC/blast counts of CML patients in chronic as well as blastic phase, it was noted that the variations in numbers of circulating leukemic cells do not correlate with the CIC levels. The significance of assessment of CIC levels in monitoring the disease in CML patients is discussed.     

Effect of 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)- quinolinone (vesnarinone) on the growth of gastric cancer cell lines.

Vesnarinone (OPC-8212; 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone) is a synthetic oral cardiotonic agent that has been used for the treatment of patients with congestive heart failure. Six days of treatment with 30 μg/ml of vesnarinone induced 20–80% growth inhibitions in five out of six gastric carcinoma cell lines examined. Cell cycle analysis revealed that the vesnarinone-sensitive TMK-1 gastric cancer cell line exhibited a significant G0–G1 arrest without evidence of apoptotic cell death induction after 48 h of treatment. Interestingly, this phenomenon was preceded by a marked reduction in the expression of cyclin A, D1 and E as well as cyclin-dependent kinase 2 (CDK2). On the other hand, no significant change was observed in the expression of p21^Waf1/Cip1, p27^Kip1 nor various growth factors and their receptor genes. Overall these results indicate that vesnarinone inhibits the growth of gastric cancer cells by down-regulating G1 cyclins and CDK2 to induce G0–G1 arrest through a pathway different from that of cyclin inactivation by p21^Waf1/Cip1 or p27^Kip1.     

Accumulation of cyclin B1, activation of cyclin B1-dependent kinase and induction of programmed cell death in human epidermoid carcinoma KB cells treated with taxol

Cyclin B1 plays a critical role in regulating cell-cycle progression from G₂ through M phase (including exit from M phase). In this study, we investigated the relationship between taxol-induced M-phase arrest, disruption of the cyclin B1-regulation pathway and apoptosis in KB cells. Continuous exposure of KB cells to 0.5 μg/ml taxol caused mitotic arrest and >90% cell death at 48 hr. Mitotic blockade peaked at 24 hr, with 68% of cells in mitosis at that time compared with 3% at baseline, and decreased thereafter. Apoptosis assessed by morphological changes and DNA ladder fragmentation was a later event, peaking at 48 hr (later time points were not studied). Taxol also caused an increase in cyclin B1 accumulation, as assessed by Western blot analysis, and stimulated cyclin B1-dependent kinase. Cyclin B1 accumulation and kinase stimulation peaked at 12 and 24 hr, respectively, at which times they were 5-fold and 90-fold higher than in control untreated cells. These effects decreased thereafter. All taxol-induced cellular effects were abrogated by the protein and RNA synthesis inhibitors cycloheximide and actinomycin D. In contrast, the endonuclease inhibitors aurintricarboxilic acid and zinc markedly inhibited taxol-induced DNA ladder fragmentation without altering taxol-induced cell-cycle arrest, cyclin B1 accumulation, activation of cyclin B1 kinase activity and cytotoxicity. We conclude that taxol-induced stimulation of cyclin B1-dependent kinase activity parallels mitotic arrest, is more pronounced than mitotic arrest and precedes the induction of programmed cell death. Int. J. Cancer 75:925–932, 1998. © 1998 Wiley-Liss, Inc.