Lymphocytic Leukemia Cell Line Research Articles

Elevated Expression of the Apoptotic Regulator Mcl-1 at the Time of Leukemic Relapse

Bcl-2, Bcl-xL, and Mcl-1 are three related intracellular polypeptides that have been implicated as negative regulators of apoptosis. In contrast, the partner protein Bax acts as a positive regulator of apoptosis. Based on the observation that all four of these polypeptides are expressed in a variety of acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) cell lines, cellular levels of these polypeptides were examined by immunoblotting in bone marrow samples harvested from 123 adult AML patients and 36 adult ALL patients before initial antileukemic therapy. Levels of Bcl-2, Mcl-1, Bcl-xL, and Bax each varied over a more than 10-fold range in different pretreatment leukemia specimens. When the 54 AML and 23 ALL samples that contained greater than 80% malignant cells were examined in greater detail, it was observed that pretreatment levels of Bcl-2 and Mcl-1 correlated with each other (R = .44, P < .001 for AML and R = .79, P < .0001 for ALL). In addition, a weak negative correlation between Bax expression and age was observed in AML samples (R = -0.35, P < .02) but not ALL samples. There was no relationship between pretreatment levels of these polypeptides and response to initial therapy. However, examination of 19 paired samples (the first harvested before chemotherapy and the second harvested 23 to 290 days later at the time of leukemic recurrence) revealed a greater than or equal to twofold increase in Mcl-1 levels in 10 of 19 pairs (7 of 15 AML and 3 of 4 ALL) at recurrence. In contrast, 2 of 19 pairs contained twofold less Mcl-1 at the time of recurrence. Approximately equal numbers of samples showed twofold increases and decreases in Bcl-2 (5 increases, 3 decreases) and Bcl-xL (1 increase, 4 decreases) at recurrence. Bax levels did not show a twofold decrease in any patient. these results, coupled with recent observations that cells overexpressing Mcl-1 are resistant to a variety of chemotherapeutic agents, raise the possibility that some chemotherapeutic regimens might select for leukemia cells with elevated levels of this particular apoptosis inhibitor.

Elevated Expression of the Apoptotic Regulator Mcl-1 at the Time of Leukemic Relapse

AbstractBcl-2, Bcl-xL, and Mcl-1 are three related intracellular polypeptides that have been implicated as negative regulators of apoptosis. In contrast, the partner protein Bax acts as a positive regulator of apoptosis. Based on the observation that all four of these polypeptides are expressed in a variety of acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) cell lines, cellular levels of these polypeptides were examined by immunoblotting in bone marrow samples harvested from 123 adult AML patients and 36 adult ALL patients before initial antileukemic therapy. Levels of Bcl-2, Mcl-1, Bcl-xL, and Bax each varied over a more than 10-fold range in different pretreatment leukemia specimens. When the 54 AML and 23 ALL samples that contained greater than 80% malignant cells were examined in greater detail, it was observed that pretreatment levels of Bcl-2 and Mcl-1 correlated with each other (R = .44,P < .001 for AML and R = .79,P < .0001 for ALL). In addition, a weak negative correlation between Bax expression and age was observed in AML samples (R = −0.35, P < .02) but not ALL samples. There was no relationship between pretreatment levels of these polypeptides and response to initial therapy. However, examination of 19 paired samples (the first harvested before chemotherapy and the second harvested 23 to 290 days later at the time of leukemic recurrence) revealed a greater than or equal to twofold increase in Mcl-1 levels in 10 of 19 pairs (7 of 15 AML and 3 of 4 ALL) at recurrence. In contrast, 2 of 19 pairs contained twofold less Mcl-1 at the time of recurrence. Approximately equal numbers of samples showed twofold increases and decreases in Bcl-2 (5 increases, 3 decreases) and Bcl-xL (1 increase, 4 decreases) at recurrence. Bax levels did not show a twofold decrease in any patient. These results, coupled with recent observations that cells overexpressing Mcl-1 are resistant to a variety of chemotherapeutic agents, raise the possibility that some chemotherapeutic regimens might select for leukemia cells with elevated levels of this particular apoptosis inhibitor.

Potentiation of 2-Chlorodeoxyadenosine Activity by Bryostatin 1 in the Resistant Chronic Lymphocytic Leukemia Cell Line (WSU-CLL): Association with Increased Ratios of dCK/5'-NT and Bax/Bcl-2

The activities of 2-chlorodeoxyadenosine (2-CdA) metabolizing enzymes, deoxycytidine kinase (dCK) and cytosolic 5'-nucleotidase (5'-NT) were measured in control and bryostatin 1 treated CLL cells using an EBV-negative WSU-CLL cell line. This cell line was established from a patient with CLL resistant to fludarabine. The results revealed a significant increase in dCK activity in bryostatin 1 treated cells at 48 and 72 h compared with the control. 5'-NT activity decreased significantly at 48 h. The ratio of dCK to 5'-NT activity was significantly increased in bryostatin 1 treated WSU-CLL cells after 48 h. WSU-CLL cells treated with bryostatin 1 exhibited an increase in the percentage of apoptotic and dead cells from control levels of 16% to 40%. This percentage was further increased to 67% following the addition of 11.2 microM 2-CdA to WSU-CLL cells pretreated with bryostatin 1. Results from Western blot analysis indicate that WSU-CLL cells express high levels of Bcl-2, Bcl-xL and c-myc, and a low level of Bax. p53 in untreated WSU-CLL cells is undetectable. WSU-CLL cells treated with bryostatin 1 showed a significant increase in the ratio of Bax to Bcl-2. To demonstrate that the bryostatin 1 mediated enhancement of 2-CdA efficacy was not restricted to in vitro cell culture, we have studied the tumor growth delay of WSU-CLL xenografts treated with placebo, bryostatin 1, 2-CdA, and bryostatin 1 followed by 2-CdA. SCID mice given bryostatin 1 at 75 microg x kg(-1) x d(-1) for 5 days followed by 30 mg x kg(-1) x d(-1) 2-CdA for 5 days in two cycles, had significantly improved tumor growth delay (P = 0.05). We conclude that bryostatin 1 is not only capable of inducing apoptosis by itself, but also sensitizes de novo resistant WSU-CLL cells to the chemo-therapeutic effects of 2-CdA. The bryostatin 1-induced increased ratio of dCK/5'-NT activity and an increased ratio of Bax/Bcl-2 are at least two mechanisms through which this natural compound is able to potentiate the anti-tumor activity of 2-CdA in otherwise resistant CLL cells.

Antineoplastic Agents 385. The Isolation and Structure of a Scalarane-Type Sesterterpene from the Indian Ocean Porifera Hyrtios erecta

Bioassay-guided separation techniques were employed to isolate (3.3·10-7% yield) a new murine P388 lymphocytic leukemia cell line inhibitor (ED50 2.9 μg/ml) 12-O-deacetyl-19-deoxyscalarin from extracts of the Republic of Maldives marine sponge Hyrtios erecta. The structure was deduced using high field 2D NMR and high resolution mass spectral methods and confirmed by X-ray crystal structure determination.

CGP 57148, a Tyrosine Kinase Inhibitor, Inhibits the Growth of Cells Expressing BCR-ABL, TEL-ABL, and TEL-PDGFR Fusion Proteins

AbstractCGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL–expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL–positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome–positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL–positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

CGP 57148, a Tyrosine Kinase Inhibitor, Inhibits the Growth of Cells Expressing BCR-ABL, TEL-ABL, and TEL-PDGFR Fusion Proteins

CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL-expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL-positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome-positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL-positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro.

The biochemical mechanism(s) by which germ cells can form specialized junctions with Sertoli cells in the seminiferous epithelium at various stages of the spermatogenic cycle is unknown. This study sought to examine the biochemical changes that are involved when germ cells are cocultured with Sertoli cells in vitro preceding the establishment of specialized Sertoli-germ cell junctions. While isolated germ cells were allowed to attach to Sertoli cells, media from both the apical and basal compartments of bicameral units were collected to assess serine and cysteine protease activity. The expression of selected serine and cysteine proteases and their corresponding inhibitors in these Sertoli-germ cell cocultures was also examined by RT-PCR. Using an [125I]-collagen film assay, a transient but significant increase in serine protease activity was noted in both the apical and basal compartments when germ cells began to settle onto the Sertoli cell monolayer preceding the formation of intercellular junctions. A specific tryptase (RNK-Tryp 2, a serine protease formerly cloned from a rat granular lymphocyte leukemia cell line, RNK-16, cDNA expression library) was shown to be expressed exclusively by Sertoli cells and not germ cells. Furthermore, Sertoli cell tryptase expression as well as urokinase plasminogen activator (u-PA, also a serine protease) increased significantly when germ cells were adhering to Sertoli cells. The decline in total serine protease activity when Sertoli-germ cell junctions were being formed was accompanied by a concomitant increase in alpha2-macroglobulin (alpha2-MG, a nonspecific protease inhibitor) expression. No significant changes in cysteine protease activity in either the apical or basal compartment were noted. However, there was a transient but significant increase in cathepsin L expression when germ cells were adhering to Sertoli cells preceding cell junction formation. The subsequent reduction in cathepsin L expression after this transient increase was accompanied by a concomitant increase in cystatin C expression. These results suggest that proteases and their corresponding inhibitors are working synergistically and are likely to be involved in the adherence of germ cells to Sertoli cells and the subsequent formation of intercellular junctions.

Comparative Activity of Melarsoprol and Arsenic Trioxide in Chronic B-Cell Leukemia Lines

Inorganic arsenic trioxide (As2O3 ) was recently shown to induce apoptosis in NB4 promyelocytic leukemic cells. We have investigated the effects of the organic arsenical, melarsoprol (a drug used for treatment of trypanosomiasis), upon induction of apoptosis in cell lines representative of chronic B-cell lymphoproliferative disorders. An Epstein-Barr virus (EBV)-transformed B-prolymphocytic cell line (JVM-2), an EBV-transformed B-cell chronic lymphocytic leukemia (B-CLL) cell line (I83CLL), and one non–EBV-transformed B-CLL cell line (WSU-CLL) were used as targets. Dose-response experiments with melarsoprol (10−7 to 10−9 mol/L) were performed over 96 hours. Unexpectedly, we found that melarsoprol caused a dose- and time-dependent inhibition of survival and growth in all three cell lines. In contrast, As2O3 at similar concentrations had no effect on either viability or growth. After 24 hours, all three cell lines treated with melarsoprol (10−7 mol/L) exhibited morphologic characteristics of apoptosis. We also observed prominent concentration-dependent downregulation of bcl-2 mRNA after 24 hours of exposure to melarsoprol in WSU-CLL, I83CLL, and JVM-2 cells. Decrease of bcl-2 protein expression was also observed in all three cell lines, whereas As2O3 had no effect on this parameter. We conclude that melarsoprol may inhibit the growth of lymphoid leukemic cell by promoting programmed cell death. Results of these studies suggest that melarsoprol shows promising therapeutic activity in these diseases, and a study to evaluate clinical effects of this drug has been initiated.

Comparative Activity of Melarsoprol and Arsenic Trioxide in Chronic B-Cell Leukemia Lines

AbstractInorganic arsenic trioxide (As2O3 ) was recently shown to induce apoptosis in NB4 promyelocytic leukemic cells. We have investigated the effects of the organic arsenical, melarsoprol (a drug used for treatment of trypanosomiasis), upon induction of apoptosis in cell lines representative of chronic B-cell lymphoproliferative disorders. An Epstein-Barr virus (EBV)-transformed B-prolymphocytic cell line (JVM-2), an EBV-transformed B-cell chronic lymphocytic leukemia (B-CLL) cell line (I83CLL), and one non–EBV-transformed B-CLL cell line (WSU-CLL) were used as targets. Dose-response experiments with melarsoprol (10−7 to 10−9 mol/L) were performed over 96 hours. Unexpectedly, we found that melarsoprol caused a dose- and time-dependent inhibition of survival and growth in all three cell lines. In contrast, As2O3 at similar concentrations had no effect on either viability or growth. After 24 hours, all three cell lines treated with melarsoprol (10−7 mol/L) exhibited morphologic characteristics of apoptosis. We also observed prominent concentration-dependent downregulation of bcl-2 mRNA after 24 hours of exposure to melarsoprol in WSU-CLL, I83CLL, and JVM-2 cells. Decrease of bcl-2 protein expression was also observed in all three cell lines, whereas As2O3 had no effect on this parameter. We conclude that melarsoprol may inhibit the growth of lymphoid leukemic cell by promoting programmed cell death. Results of these studies suggest that melarsoprol shows promising therapeutic activity in these diseases, and a study to evaluate clinical effects of this drug has been initiated.

Inhibition of Apoptosis in a Human Pre-B–Cell Line by CD23 Is Mediated Via a Novel Receptor

Human CD23 is a 45-kD type II membrane glycoprotein, which functions as a low-affinity receptor for IgE and as a ligand for the CD21 and CD11b/CD11c differentiation antigens. CD23 is released from the surface of cells as soluble fragments, and a 25-kD species of soluble CD23 (sCD23) appears to act as a multifunctional cytokine. In this report, sCD23 is shown to sustain the growth of low cell density cultures of a human pre-B–acute lymphocytic leukemia cell line, SMS-SB: no other cytokine tested was able to induce this effect. Flow cytometric analysis indicates that sCD23 acts to prevent apoptosis of SMS-SB cells. SMS-SB cells cultured at low cell density possess low levels of bcl-2 protein. Addition of sCD23 to cells at low cell density maintained bcl-2 expression at levels equivalent to those observed in SMS-SB cells cultured at higher cell densities. No CD23 mRNA was found in SMS-SB cells, ruling out an autocrine function for CD23 in this cell line model. Although SMS-SB cells do not express the known receptors for CD23, namely CD21, CD11b-CD18, or CD11c-CD18, the cells specifically bind CD23-containing liposomes, but not glycophorin-containing liposomes. Binding of CD23-containing liposomes is inhibited by anti-CD23 but not by anti-CD21 or anti-CD11b/c monoclonal antibodies. The data show that sCD23 prevents apoptosis of the SMS-SB cell line by acting through a novel receptor.

Inhibition of Apoptosis in a Human Pre-B–Cell Line by CD23 Is Mediated Via a Novel Receptor

AbstractHuman CD23 is a 45-kD type II membrane glycoprotein, which functions as a low-affinity receptor for IgE and as a ligand for the CD21 and CD11b/CD11c differentiation antigens. CD23 is released from the surface of cells as soluble fragments, and a 25-kD species of soluble CD23 (sCD23) appears to act as a multifunctional cytokine. In this report, sCD23 is shown to sustain the growth of low cell density cultures of a human pre-B–acute lymphocytic leukemia cell line, SMS-SB: no other cytokine tested was able to induce this effect. Flow cytometric analysis indicates that sCD23 acts to prevent apoptosis of SMS-SB cells. SMS-SB cells cultured at low cell density possess low levels of bcl-2 protein. Addition of sCD23 to cells at low cell density maintained bcl-2 expression at levels equivalent to those observed in SMS-SB cells cultured at higher cell densities. No CD23 mRNA was found in SMS-SB cells, ruling out an autocrine function for CD23 in this cell line model. Although SMS-SB cells do not express the known receptors for CD23, namely CD21, CD11b-CD18, or CD11c-CD18, the cells specifically bind CD23-containing liposomes, but not glycophorin-containing liposomes. Binding of CD23-containing liposomes is inhibited by anti-CD23 but not by anti-CD21 or anti-CD11b/c monoclonal antibodies. The data show that sCD23 prevents apoptosis of the SMS-SB cell line by acting through a novel receptor.

Possible Roles of an Adult T-Cell Leukemia (ATL)-Derived Factor/Thioredoxin in the Drug Resistance of ATL to Adriamycin

AbstractChemotherapy for adult T-cell leukemia (ATL) has been reported to fail to induce complete remission because of drug resistance in most patients. We have examined the expression of an ATL-derived factor (ADF)/thioredoxin in relation to resistance to adriamycin (ADM) in various T-cell leukemia cell lines including ATL cell lines. Immunoblot analysis demonstrated that ATL cell lines expressed ADF/thioredoxin at levels 2.8 to 12 times those of other T-cell acute lymphocytic leukemia (T-ALL) cell lines, and that ATL cell lines were 2 to 15 times more resistant to ADM than other T-ALL cell lines. Therefore, we established ADM-resistant cell lines from three different ATL cell lines, and examined the correlation between ADM resistance and expression of ADF/thioredoxin. ADM-resistant ATL cell lines were also found to be resistant to other drugs such as cisplatin and etoposide, and they expressed ADF/thioredoxin at levels 5 to 10 times those of parent ATL cell lines. Diamide and sodium selenite, which have been reported to inhibit ADF/thioredoxin, restored the sensitivity to ADM in ATL and ADM-resistant ATL cell lines. The MDR-1 gene product, a membrane P-glycoprotein (Pgp), was not expressed on ATL cell lines or ADM-resistant ATL cell lines. Topoisomerase II and glutathione peroxidase activities in T-cell leukemia cell lines were not correlated with ADM resistance. These results suggest that ADF/thioredoxin may play an important role in the drug resistance of ATL cells to ADM.

Ligation of MHC class I induces apoptosis in human pre-B cell lines, in promyelocytic cell lines and in CD40-stimulated mature B cells.

A murine mAb (BAL-1) was previously shown to induce apoptosis when cross-linked on the cell surface of different B acute lymphocytic leukemia (ALL) and pro-myelocytic cell lines. The present study shows that BAL-1 specifically recognizes the MHC class I (MHC-I). The apoptotic response was not dependent on the epitope specificity, since other anti-MHC-I antibodies, reacting with different monomorphic determinants of the alpha chain or beta 2-microglobulin, also induced apoptosis in these cells. However, external cross-linking of antibodies was strictly required for the apoptotic effect. Among cells originating from mature peripheral blood B cells, anti-CD40-stimulated cells were susceptible to anti-MHC-I-induced apoptosis, whereas B cells activated with Staphylococcus aureus Cowan I (SAC) or with the superantigen staphylococcal enterotoxin A (SEA) were non-responsive. Mature SEA-activated T cells were also resistant to MHC-I-induced apoptosis. In situ terminal deoxynucleotidyl transferase staining of apoptotic cells at various stages during MHC-I-induced cell death revealed that apoptosis occurred predominantly in the G2/M phase of the cell cycle, with the first apoptotic cells appearing after approximately 12 h of incubation. These results suggest a role for MHC-I-mediated apoptosis during differentiation and activation of certain hematopoietic cells.

A DNA polymerase α accessory protein exhibits structural and functional similarities to SV40 large tumor antigen

Untransformed cells have been proposed to require a protein homologous to SV40 large tumor antigen (TAg) which functions as a component of the replicase complex during the initiation of DNA synthesis. By definition, this should be a phosphoprotein which interacts with the retinoblastoma protein (pRb) in G 0 or early G 1, and is capable of binding to and potentiating the activity of DNA polymerase α (pol α). This protein should also be an ATP-dependent helicase which interacts with the single-stranded DNA (ssDNA) binding protein, RP-A. Because of these requirements, a TAg homologous protein could be expected to contain epitopes with amino acid sequences similar to those of TAg at critical functional sites, such as ATP, pRb and pol α binding sites. TAg and a putative cellular homolog of TAg, DNA pol α accessory protein (αAP), were compared for pRb and pol α interaction, and for immunological identity. The analyses utilized immunoaffinity-purified TAg and pRb from a baculovirus expression system, and DNA pol α/primase and αAP chromatographically isolated from a mouse lymphocytic leukemia cell line. Monoclonal antibodies specific for the pol α or pRb binding sites on TAg interacted with αAP strongly enough to be employed for immunoaffinity purification of αAP. Anti-pRb and anti-TAg reciprocally coimmunoprecipitated pRb bound to TAg and pRb bound to αAP. The functional consequences of pol α interaction with TAg or αAP in the presence or absence of pRb was determined using pol α nucleotide incorporation assays. αAP exhibited the capacity to stimulate pol α activity, a capacity which was diminished in the presence of pRb. Lastly, TAg and αAP independently co-purified with pol α through a multi-step chromatographic protocol. These data indicate that a pol α accessory protein, αAP, exhibits functional and immunological similarities to SV40 TAg, suggest that αAP is involved in regulation of the initiation of DNA synthesis, and support the proposal that αAP may be a normal cell protein homologous to SV40 large T antigen.

Human T-Cell Leukemia Virus-1 Encoded Tax Protein Transactivates α1→3 Fucosyltransferase Fuc-T VII, Which Synthesizes Sialyl Lewis X, a Selectin Ligand Expressed on Adult T-Cell Leukemia Cells

Leukemia cells in patients with adult T-cell leukemia (ATL) and related cell lines strongly express the carbohydrate determinant sialyl Lewis X, a ligand for selectins. Its expression is thought to be related closely to the extravascular infiltration of the leukemia cells. Human T-cell leukemia virus type 1 (HTLV-1), the etiological agent of ATL, produces Tax protein, which is implicated in leukemogenesis through its transactivating effect on various cellular genes. In this study we investigated the transactivating effect of HTLV-1 Tax on the α1→3 fucosyltransferase Fuc-T VII, the putative rate-limiting enzyme in the synthesis of sialyl Lewis X in human leukocytes using JPX-9 cells. JPX-9 is a subclone of a non-ATL human lymphocytic leukemia cell line, Jurkat, and was established by introducing a metallothionein promoter-driven Tax expression plasmid. The JPX-9 cells as well as parental Jurkat cells did not express Fuc-T VII mRNA under normal culture conditions. When cultured in the presence of 10 μM CdCl2, Tax was induced and a significant amount of the Fuc-T VII message was ascertained by Northern blotting. The amount of the message was 24.5 times as much as was detected in non-treated cells, and was comparable to that which appeared by TPA stimulation of the cells, which is supposed to simulate the sequence of events occurring in normal activation of T lymphocytes activated by more physiological stimuli. Sialyl Lewis X determinant was expressed at the surface of CdCl2-treated cells, while the determinant was not detectable on either unstimulated JPX-9 or parental Jurkat cells. These results indicate that expression of sialyl Lewis X on leukemic cells in patients with ATL is at least partly due to the transactivation of the Fuc-T VII gene induced by the HTLV-1 Tax, and suggest that this leads to the accelerated extravascular infiltration of ATL cells.

Basic fibroblast growth factor (bFGF) upregulates the expression of bcl-2 in B cell chronic lymphocytic leukemia cell lines resulting in delaying apoptosis.

Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine which has recently been shown to delay fludarabine-induced apoptosis in B cell chronic lymphocytic leukemia (B-CLL) cells. To investigate the potential mechanism of bFGF-mediated delay of apoptosis, two EBV-transformed B prolymphocytic cell lines (JVM-2, JVM-13), one EBV-transformed B-CLL cell line (I83CLL), and one non-EBV-transformed B-CLL cell line (WSU-CLL) were used as a model for chronic lymphoid malignancies. Viability data of cells treated with fludarabine alone or in combination with bFGF demonstrated that the addition of bFGF to the cells resulted in prolonged survival. Quantitative assessment of apoptosis-associated DNA strand breaks by in situ TdT labeling showed a protective effect of bFGF on fludarabine-treated cells. The potential effect of bFGF on bcl-2 mRNA expression was analyzed by Northern blotting. Stimulation with bFGF led to a time-dependent accumulation of bcl-2 specific mRNA in all three cell lines. Maximal levels of bcl-2 mRNA expression were detected after 8 h in JVM-2, and after 18 h in JVM-13 and I83CLL. Intracellular bcl-2 protein was also found to be increased upon bFGF stimulation in both EBV- and non-EBV-transformed cells. In addition, exposure of cells from three patients with B-CLL to bFGF showed an upregulation of bcl-2 protein after 4-8 h. Our data demonstrate that bFGF upregulates the expression of bcl-2 in these cells, suggesting that this increase in bcl-2 expression may play a role in the delay of fludarabine-induced apoptosis.

The bcl-2 Gene Status of Human Head and Neck Cancer Cell Lines

The bcl-2 oncogene was originally found in the translocation in a pre-B cell acute lymphocytic leukemia cell line. Since then a high expression of Bcl-2 has been found in many types of cancer. The bcl-2 gene encodes an intracellular membrane-associated protein. Overexpression of bcl-2 inhibits apoptosis induced by many drugs and radiation. In this study the bcl-2 gene status of 9 human head and neck squamous cell carcinoma cell lines was studied. Mutations of the bcl-2 gene were studied at mRNA and DNA levels. The presence and abundance of the Bcl-2 protein in cells were also investigated. In earlier studies the p53 tumour suppressor gene was screened for point mutations, and the radiosensitivity of these cell lines was measured. We were able to amplify bcl-2 cDNA from 5 of the 9 cell lines, which shows that bcl-2 was expressed in these cells. No point mutations were found in the bcl-2 gene in any of these cell lines. Loss of heterozygosity was observed in 2 cell lines at the bcl-2 locus, and these cell lines had no detectable levels of bcl-2 mRNA or Bcl-2 protein. The Bcl-2 protein was abundant in the cell lines with the wild-type p53 gene, and these cell lines were radioresistant. The Bcl-2 protein was also found in many other cell lines in mitotic cells. It seems that cells expressing bcl-2 are radioresistant, and even functional p53 cannot induce apoptosis in these cells.

Microtubule-dependent multilobular organization of the nucleus in sensitive and multidrug-resistant L0 leukemia cells.

The relationship between the nuclear morphology and the microtubular organization of L100 and L1000 cells, two vincristine-induced multidrug resistant human acute lymphocytic leukemia cell lines, was examined and compared to that of L0 parental cells. The L0 parental cells contained a round nucleus and the microtubules were evenly distributed throughout the cytoplasm. In contrast, the microtubules of the L100 and L1000 cells were localized between the lobular structures of a multilobulated nucleus. Disassembly of microtubules in L100 and L1000 cells by colchicine resulted in the loss of the multilobulated morphology of the nucleus. While the total cellular content of tubulin of L0 and L100 cells was similar, the content of microtubules of L100 cells was only 55% of that observed in L0 cells. Two, 28 kDa (pI 6.9) and 31 kDa (pI 4.4), microtubule-associated proteins were found to be overexpressed in L100 and L1000 cells. The results indicate that the multilobulated nuclear morphology of L100 and L1000 cells is dependent upon the unique and intact organization of the microtubules; the distinct organization of the microtubules and the multilobular nuclear morphology of the two resistant cell lines may be due to the differential expression of specific microtubule-associated proteins.

Synthetic Approaches to the Angucycline Antibiotics: A Concise Entry to the Ring System of PD 116740 and TAN 1085

The angucycline antibiotics have attracted a great deal of attention in the past decade due to their interesting biological properties.1 Although several procedures for the synthesis of individual angucyclines have been reported,2 an efficient general asymmetric method has yet to be developed. We have recently reported a synthesis of the racemic forms of rubiginone B1 (1) and B2 (2), emycin A (3), and ochromycinone (4),2i,j,m which were synthesized from the common intermediate (()-5a formed from the highly stereoselective tetra-O-acetyl diborate promoted Diels-Alder reaction of juglone (5hydroxy-1,4-naphthoquinone) and the diene (()-6a. In an extension of this work, the reaction of juglone with diene (()-6b promoted by a Lewis acid derived from (S)(+)-3,3′-diphenyl-1,1′-binaphthalene-2,2′-diol and BH3‚ THF gave cycloadduct (+)-5b (ee >98%). Modification of (+)-5b resulted in the syntheses of 3 and 4 in enantiomerically pure form.3 In an attempt to extend the versatility of this DielsAlder strategy our attention turned toward angucyclines possessing aromatic A ring and hydroaromatic B ring functionality. We felt that intermediates similar to (+)5b could serve as precursors to PD 1167404 (7), which has been shown to exhibit in vitro activity against L1210 lymphocytic leukaemia and HCT-8 human colon adenocarcinoma cell lines, and the aglycon of TAN 10855 (8), which shows angiogenesis inhibition. The easily accessible Diels-Alder cycloadduct (()-92m would serve as an appropriate model to ascertain the viability of our approach. Our synthetic strategy is outlined in Scheme 1. We felt protected trans diol functionality at C-5 and C-6 could be introduced by epoxidation of the C-4a-C-5 double bond. Examination of the crystal structure of a closely related cycloadduct3 indicated that oxidation would occur from the face of the alkene anti to the C-6 methoxyl group. A neutral oxidant would be required as cycloadduct 9 is unstable under

Antineoplastic Agents, 325. Isolation and Structure of the Human Cancer Cell Growth Inhibitory Cyclic Octapeptides Phakellistatin 10 and 11 from Phakellia sp.

The two new marine sponge (Phakellia sp., western Pacific Ocean) constituents, phakellistatin 10 [1] and 11 [2], were found to be cyclic octapeptides that significantly inhibited growth of the murine P-388 lymphocytic leukemia (ED50 values of 2.1 and 0.20 micrograms/ml, respectively) and human cancer cell lines. The structures were established based on results of extensive tandem ms/ms and high-field (500-MHz) 2D 1H- and 13C-nmr analyses. All of the amino acid units (except Trp, not determined) were found to correspond to the (S)-configuration.