Bryostatin 1 induces apoptosis and augments inhibitory effects of vincristine in human diffuse large cell lymphoma

Leukmna

Pergamon

Research Vol. I’), No. 9, pp. 667-673, 1995. Copyright 0 1995 Elscvier Science Ltd Printed in Great Britain. All rights reserved 0145-2126/95 $9.50 + 0.00

01452126(95)00037-2

BRYOSTATIN 1 INDUCES APOPTOSIS AND AUGMENTS INHIBITORY EFFECTS OF VINCRISTINE IN HUMAN DIFFUSE LARGE CELL LYMPHOMA Ramzi M. Mohammad,* Hariharan Diwakaran,* Auday Maki,* Mohamed A. Emara,? George. R. Pettit,‘j Bruce Redman* and Ayad Al-Katib* *Division of Hematology and Oncology, Department of Internal Medicine, Wayne State University, P.O. Box 02143, Detroit, MI 48201, U.S.A.; THenry Ford Hospital, Detroit, Michigan, U.S.A.; and ZCancer Research Institute, Arizona State University, Tempe, Arizona, U.S.A. (Received 7 December 1994. Accepted 16 February 1995) Abstract-Bryostatin 1 (Bryol), a macrocyclic lactone and a protein kinase C activator, is isolated from the marine bryozoan Bugula neririna. In this study we describe its effect, alone or after sequential use with vincristine (VCR), on the human diffuse large cell lymphoma cell line WSU-DLCL2. Our results show that both Bryol and VCR induced apoptosis as demonstrated by morphological examination, DNA flow cytometry (FCM), and DNA fragmentation on agarose gel electrophoresis. Cells pretreated for 24 h with Bryol and then exposed to VCR showed an increase in apoptosis compared to cells that were exposed to Bryol or VCR alone. We also studied the effects of Bryol, VCR and their combination on cell growth, bcl-2 and p53 expression, and inhibition of cell proliferation as measured by [3Hl-thymidine incorporation. Cell analysis showed significant growth inhibition of WSU-DLCL2 cells by the Bryol/VCR combination as compared to either agent alone. lmmunocytochemistry (ICC) revealed that relative bcl-2 oncoprotein expression was decreased in cells treated with Bryol, or VCR separately and was abolished by combining both drugs. When examined by ICC, WSU-DLCLS cells were initially negative for the p53 protein. However, upon treatment with the above agents, the relative expression of p53 was moderate on Bryol-or VCR-treated cells and strong on cells treated with the Bryol/VCR combination. Cell proliferation as measured by f3Hlthymidine incorporation revealed significant inhibition of tumor growth by exposure to the agents when compared to the control. In contrast, Bryol, VCR and their combination did not show any inhibition of normal bone marrow growth. These findings taken together, suggest that the exposure of WSU-DLCL2 cells to Bryol prior to treatment with VCR enhances apoptosis, a phenomenon which might be exploited for future therapies. Key words: B-cell, immunocytochemistry,

diffuse large cell lymphoma, bryostatin 1, vincristine.

apoptosis,

bcl-2,

~53, t3H]-thymidine,

developed. One attempt to maximize the effect of chemotherapeutic agents and possibly achieve cures in presently incurable tumors, is to combine cytotoxic agents with biological agents that have a different mode of action. Programmed cell death or apoptosis has been recognized as a fundamental tissue homeostatic mechanism within a wide range of physiological and pathological conditions, including cancer. Bryostatin 1 (Bryol), a macrocyclic lactone, is a potent activator of protein kinase C [S, 61 and has antitumor, immune modulating and differentiation capacity on a number of B-cell lymphomas and leukemias [6-81. The vinca alkaloid vincristine (VCR) is widely used, both experimentally as a stathmokinetic agent, and clinically

Introduction Non-Hodgkin’s lymphomas are primarily a group of Bmalignancies. According to the International Working Formulation, diffuse large cell non-Hodgkin’s lymphomas (DLCL) are classified as intermediate grade lymphomas [l]. Using standard chemotherapy with CHOP, only a third of patients achieve long-term survival [2]. A number of other regimens have been tried, but none were found to be superior to CHOP [3,4]. Thus, it is important that alternative regimens be Correspondence to: Professor Ramzi M. Mohammad, Wayne State University School of Medicine, P.O. Box 02143, Detroit, MI 48201, U.S.A. (Tel: 313 577 7919; Fax: 313 577 7925). 667

R. M. Mohammade/ trl

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in the treatment of a variety of human cancers [9, lo]. VCR binds to the microtubular protein tubulin, resulting in disruption of the mitotic spindle apparatus in metaphase [lo, 111 and has also been shown to cause apoptosis of interphase cells in human Burkitt’s lymphoma [12]. In this study, we investigated the effects of these agents on an intermediate grade lymphoma cell line (WSU-DLCL2) [13]. We present evidence, based on morphological, DNA fragmentation on agarose gel electrophoresis, flow cytometric and immunocytochemistry examination, that Bryol induces apoptosis and potentiates the inhibitory effects of vincristine. Such interaction is associated with modulation of bcl2 and p53 proteins. Information obtained from this study might be useful in guiding the clinical application of Bryol, a promising agent that is entering Phase I clinical trials in relapsed lymphomas and chronic lymphocytic leukemia at our institute. Materials and Methods Cell culture

The malignant human cell line WSU-DLCL2 (diffuse large cell lymphoma) established in our laboratory was used in this study [ 131. This cell line grows in liquid culture consisting of RPMl-1640 supplemented with 1% glutamine (Gibco BRL, Gaithersburg, MD, U.S.A.), 10% (v/v) heat inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT, U.S.A.), penicillin (100 pgiml), and streptomycin (100 @ml). Cells were seeded at a concentration of 1.5 x lO”/ml in a 24 well plate and incubated at 37°C in a 5% CO2 humidified incubator after adding the drugs in the concentrations and manner described below. Bone marrow was obtained from human ribs. The ribs were split lengthwise so that the marrow was exposed, then the marrow was flushed out with RPM1 1640 medium using a 23G needle and a 5 ml syringe. Mononuclear cells were then isolated using Ficoll-Hypaque density sedimentation, washed and viability by Trypan Blue exclusion was determined. Cell density was adjusted to l&/ml and cultured as previously described [ 141. Drugs and reagents

The total cell number as well as viability were obtained daily for 5 days on all wells using Trypan Blue (0.4%) exclusion (Gibco, NY, U.S.A.). Mel-phologic

features

of apoptosis

For light microscopic examination, cytocentrifuge smears from all cultures were prepared daily for 5 days, using the Cytospin 2 centrifuge (Shandon Southern Instruments, Sewickley, PA, U.S.A.). The smears were air dried, stained with tetrachrome for 5 min, and examined with a Nikon Labophot microscope (Nikon, Garden City, NY, U.S.A.). Five different fields of the stained cytocentrifuge smear were counted for viable, mitotic, apoptotic and dead cells. At least 50 cells per field were counted, presented as a percentage of the control, and the data were statistically analyzed. Features of apoptosis that were looked for included cell shrinkage, nuclear chromatin condensation, formation of membrane blebs and apoptotic bodies. Features of cell death included cell swelling, nuclear expansion and gross cytolysis. Cell proliferation

by [‘HI-thymidine

incorporation

Cells were cultured in 96 well round bottom plates at 5 x lo4 cells per well. Bryol (200 nM), VCR (0.05 @ml), both (200 nM+0.05 ng/mI) or nothing (control) was added to the wells. All conditions were in triplicate. Plates were incubated at 37°C in 5% COz for 4 days. Wells were then pulsed with 1 pCi/well of [“HI-thymidine 18 h before harvesting. Thymidine uptake in control and treated cultures was determined by beta counter. The percentage suppression of proliferation by the agents was calculated from the formula: 1 - [c.p.m. of treated cells/c.p.m. of control cells] X 100. of DNA fragmentation by agarose gel electrophoresis DNA was extracted from cells treated with Bryol, VCR, Bryol/VCR combination, and control cultures using 10 mM Tris HClil mM EDTA, pH 8.0 (TE), containing 0.2% Triton X-100. DNA was precipitated from the lysate by 0.1 vol 5 M sodium acetate (pH 5.0) and 3 vol 95% ethanol. After centrifugation, DNA pellets were air dried and resuspended in 25 pl of TE containing 0.1% SDS. Loading buffer was added to the samples at 1:5 (v/v) ratio and the samples were incubated for 10 min at 6O”C, followed by agarose gel (0.75%) electrophoresis in Tris borate buffer (45 mM Tris borate/l mM EDTA, pH 8.0). DNA was visualized with ethidium bromide under UV light and photographed.

Analysis

Bryol [dissolved in dimethyl sulphoxide (Fisher Scientific Co., Fair Lawn, NJ, U.S.A.) at 2 PM] was directly diluted in phosphate buffered saline (PBS) to yield a final concentration of 200 nM, as previously described [S]. This concentration was shown to have a differentiation effect on several B-cell tumors [15, 161. VCR [obtained from Sigma (St. Louis, MO, U.S.A.)] was used at a concentration of 0.05 @ml. For the Bryol/VCR combination, Bryol was added 24 h prior to adding VCR. The cells were examined 24 h after VCR exposure [16] and compared to control, Bryol- and VCR-treated cells at the same time points. Cell growth

analysis

WSU-DLCL2 cells were seeded in 24 well culture plates (Costar, Cambridge, MA, U.S.A.) at a concentration of 1.5 x lo5 viable cells per ml. Untreated (control), Bryol-, VCR-, and Bryol/VCR-treated cultures were set in duplicate. Incubation of cultures was at 37-C, 5% CO* in a humidified incubator.

-

01

. 0

I 1

.

I 2

.

1

f

3

4

Days Fig. 1. Effects of Bryol (200 nM), VCR (0.05 @ml), Bryo l/ VCR combination on the growth of WSU-DLCL2 cells.

Bryostatin 1-apoptosis

669

induction and augmentatior of inhibitory effects

Table 1. The effect of Bryol, VCR and their combination on WSU-DLCL2 and normal bone marrow (nbm) cells Treatment*

[‘H]-Thymidine incorporation [c.pm. i S.D. (% suppression)] WSU-DLCL2 cells nbm

Control Bryol ‘VCR BryoliVCR

4737*353t 2901 k 335 (39): 3322 + 48 (30)$ 2733 k l-46 (42);

5134k268.t 4173+ 1009 (19)“” 5643 k 354 (00) 6712 + 534 (00)

*Cells were cultured (5 x 103/well) for 4 days with or without agent (10 ~1110~ cells) at the onset of cultures. SControl: proliferation in the absence of agents, measured on day 4. suppression in cell proliferation by agent relative to controls were P
Flow cytometric detection of apoptosis For flow cytometric detection of apoptosis, 10h cells from each of the different treatments were fixed in absolute alcohol for 30 min at 4°C. After treatment with RNase for 40 min at 37°C cells were stained with propidium iodide (50 ugiml) and analyzed by FCM or FACS scan as previously described [S]. Apoptotic cells are identified on DNA histograms as a peak in the hypodiploid region.

thereafter.

This

the indicated spontaneous ZPercentage significant at

arrest

was

statistically

significant

(P
Analysis of bcl-2 and ~53 oncoprotein expression by immunocytochemistry Cytocentrifuge smears on round coverslips were prepared from all treatment groups using a Cytospin 2 centrifuge (Shandon Southern Instruments, Sewickley, PA, U.S.A.). Smears were fixed with 95% ethanol and 5% glacial acetic acid for 30 min at room temperature. Cells were then washed with 0.1 M PBS and incubated overnight with mouse monoclonal anti-human 1~~1-2(Dako Co., CA, U.S.A.) and mouse anti-human ~53 antibodies (Oncogenes Science, NY, U.S.A.) at a dilution of 1:60 and 1:25, respectively. The cells were washed extensively with PBS and incubated for 2 h with goat anti-mouse antibodies conjugated to alkaline phosphatase. A subsequent enzyme catalyzed color reaction with 5-bromo4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) was used to visualize the protein. The whole experiment was repeated three times, and slides were independently examined by more than one investigator using the same scale (- negative; + weak; ++ moderate; +++ strong expression). The data presented for bcl-2 and ~53 are the mean of three readings.

Results Cell proliferation

The effects of Bryol, VCR, and their combination on the growth of WSU-DLCL2 cells is shown in Fig. 1. Bryol caused growth inhibition (arrest) during the first 48 h of exposure. After that, cells showed some signs of proliferation recovery. VCR caused moderate growth inhibition. Adding Bryol 24 h before VCR caused greater inhibition than either agent alone. Complete growth inhibition was noticed with the Bryol/VCR combination, and no signs of growth recovery were seen

Fig. 2. Photograph showing the characteristic features of apoptosis in WSU-DLCL2 cells (x 1000). (A) WSU-DLCL2 cells treated with Bryol showing apoptosis with characteristic apoptotic bodies and (B) cells treated with VCR showing arrest in mitosis.

670

R. M. Mohammad

et ul.

n Viable q Mitosis El Apoptosis El Dead

”

Control

Bryol

B/V

VCR

Fig. 3. Bar-chart showing the percentage of viable, mitotic, apoptotic and dead cells in control, Bryol, VCR and Bryoli VCR treated WSU-DLCL2 cells.

that Bryol, VCR and their combination caused significant (PcO.05) inhibition in WSU-DLCL2 cell proliferation (Table 1). In contrast, Bryol, VCR and Bryol/VCR combination showed no significant inhibitory effect on normal human bone marrow in the concentrations used (Table 1).

cells and was higher (P
Apoptosis and mitosis WSU-DLCL2 cells were assessed morphologically for apoptosis and mitosis. Features of apoptosis that were looked for included cell shrinkage, nuclear condensation and apoptotic bodies. The treated cells showed characteristic features of apoptosis and mitosis (Fig. 2A, B). Bryol induced apoptosis in 23% of the cells and VCR in 31%; both were statistically significant as compared with the control (P
199. !. PI-AREA

3999. 30.

199. I.

E”* 4

WV

V

60

120

180

240

60

120

180

240

Channel

60

120

180

240

Number

Fig. 4. DNA flow cytometry of WSU-DLCL2 cells showing apoptotic peaks (AP) in different treatments. C = control, B = Bryol, V = VCR, B/V = Bryol/VCR combination.

60

120

180

240

Bryostatin 1-apoptosis

671

induction and augmentation of inhibitory effects

Table 2. lmmunocytochemistry studies for bc12 and p53 oncoprotein expression on treated and control WSU-DLCL2 cells

12345

Agent

bcl2

Control Bryol VCR Bryol/VCR

ttt t t -

- negative; t weak; tt moderate; ttt

Fig. 5. Classical laddering pattern caused by DNA fragmentation on agarose gel electrophoresis induced by the drugs. 1 = DNA mass ladder supplied by Gibco BRL, range 100-800 bp, 2 = control, 3 = Bryol, 4 = VCR, 5 = Bryol/VCR combination.

laddering pattern resulting from the generation of multiple nucleosome fragments was seen, indicating that these cells underwent apoptosis. Products expression of hcl-2 and ~53 genes The relative expression of the oncoprotein bcl-2 and the tumor-suppressor protein p53 as examined by immunocytochemistry are shown in Table 2. The untreated (control) cells strongly expressed the bcl-2 oncoprotein. In contrast, bcl-2 exhibited weak expression in cells treated with either Bryol, or VCR, separately, but was abolished by the Bryol/VCR combination. The untreated WSU-DLCL2 cells did not express ~53. However, upon treatment with either Bryol, or VCR, the cells showed a relatively higher expression of ~53. The combination of Bryol/VCR resulted in strong p53 expression.

Discussion Our data indicates that Bryostatin 1 induces apoptosis, and when used in combination with vincristine, it augments the inhibitory effects of VCR administered by itself in a human diffuse large cell lymphoma cell line. This increased effect of the Bryol/VCR combination was associated with down-regulation of bcl-2 oncoprotein and increased expression of the p53 protein. The apoptotic effect induced by Bryol, VCR and their combination was observed by morphology, flow cytometric analysis and by the observation of a DNA ladder

P53 tt tt ttt strong expression

on agarose gel electrophoresis (Figs 2, 4 and 5). In the past, it has been shown that VCR induces apoptosis in the human Burkitt’s lymphoma cell line BM13674 [12] and that Bryol also potentiates the antileukemic action of l-(0-D-arabinofuranosyl)-cytosine (Ara-C) on HL-60 human promyelocytic leukemia cells via apoptosis [17]. Presently, it is not clear how Bryol induces and potentiates apoptosis. Apoptosis is an active, energy dependent process which depends on the expression of certain genes [18]. Among them, bcl-2 is known to inhibit apoptosis [19-211 and the tumor suppressor gene (wild type) ~53 induces apoptosis [22,23]. Our results have shown that Bryol, by itself, can reduce bcl-2 expression in WSU-DLCL2 cells and can act with VCR in reducing bcl-2 expression further. These findings have been associated with a proportionate increase in ~53 expression (Table 2). An inverse correlation has been found between the expression of bcl-2 and p53 oncoproteins in human breast cancer cell lines [24]. This inverse correlation may be a key factor in the initiation of apoptosis. The changes in the expression of these oncogenes [25,26] may initiate endonuclease activity which in turn acts on chromatin and produces the DNA fragmentation seen on electrophoretic gels. Thus, any therapy that decreases bcl-2 expression and/or increases p53 expression may be expected to increase cell death by increasing apoptosis. The number of apoptotic cells in the Bryol/VCR combination increased when compared with either agent given alone (Figs 3 and 4) but the number of cells in mitosis decreased dramatically when compared with VCR alone. This effect could be due to the differentiation of WSU-DLCL2 cells exposed to Bryol leading to accumulation in GO/G1 and apoptosis. Cells that escape this effect and progress in the cell cycle passed GO/G1 will be held in mitosis and undergo apoptosis by VCR [lo, 111. Bryol may be acting by several mechanisms to induce apoptosis and augment the effect of cytotoxic agents. First, Bryol may inhibit bcl-2 expression and/or increase p53 expression by an unexplained mechanism. Second, PKC activators like Bryol and the cytotoxic agent ara-C have also been shown to induce upregulation of certain oncogenes like c-jun and c-fos

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Mohammad

known to be associated with initiation of endonucleolytic DNA cleavage and apoptosis [27-291. Third, it may act by down-regulating MDR gene expression and thus increase susceptibility to the cytotoxic agents [30]. Bryol showed a temporary proliferation inhibitory effect on WSU-DLCL2 cells. The effect was most evident during the first 48 h of the incubation period. Growth recovery, however, did not eliminate the gap between control and Bryol-treated cultures (Fig. 1). Similar results were seen on different non-Hodgkin’s cell lines in vitro [8]. On the other hand, in the Bryol/ VCR combination culture there was no increase in cell number in the first 24 h, followed by a decrease in the subsequent 48 h. Since the [“HI-thymidine incorporation assay showed only suppression in cell proliferation with Bryol, VCR and their combination, compared with control (PC O.OS), we conclude that the combined effects of the drugs on the cell cycle and increased apoptosis lead to a net decrease in cell growth relative to each agent alone. The agents alone or in combination showed no significant suppression of normal human bone marrow cells (Table 1). Recognition that Bryol induces apoptosis and potentiates the cytotoxic action of VCR, in addition to several other modes of action, may have important clinical implications. Understanding the interactions between Bryol and standard chemotherapy agents could guide the development of new therapeutic regimens and lead to an improved treatment outcome in B-cell tumors in man. Acknowledgemerzts-This work was made possible by a grant from the Elsa U. Pardee Foundation and by the Al-Katib Cancer Research Fund of Harper Hospital. Flow cytometry was performed at the Ben Kasle flow cytometry facility of the Meyer L. Prentis Comprehensive Cancer Center of Metropolitan Detroit supported by USDHHS CA 22453.

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