Mitogen-activated protein kinase is required for bryostatin 1-induced differentiation of the human acute lymphoblastic leukemia cell line Reh.

Bryostatin 1 (bryo 1) is known to induce the differentiation and cell cycle arrest of human lymphoid leukemia cells in vitro. The extracellular signal-regulated kinase (ERK), originally identified as a participant in mitogenic signaling, has recently been implicated in the signaling of cellular differentiation. To examine the role of the ERK/mitogen-activated protein (MAP) kinase pathway in B-lymphoid cell differentiation of the Reh Acute Lymphoblastic Leukemia cell line, the effects of bryo 1 on ERK activation were determined. On bryo 1 treatment, the activity of ERK2 (p42) rapidly increased, with ERK1 (p44) protein levels remaining constant. p44/42 immunoprecipitates from lysates of bryo 1-treated cells had increased their ability to phosphorylate the transcription factor Elk-1. Constitutive AP-1 activity was shown to be potentiated after bryo 1 treatment using electrophoretic mobility shift assays. The protein composition of the AP-1 transcription factor complex activated by bryo 1 was analyzed using supershift analysis with specific antibodies against c-Fos, Fos B, c-Jun, Jun B, and Jun D proteins. Supershift analysis revealed that the bryo 1-induced AP-1 complex was composed predominantly of Fos B and Jun D. Therefore, we evaluated the effects of inhibiting MAP/ERK kinase (MEK) on both DNA binding and cellular differentiation. Treatment of Reh cells with 20 microM PD98059, a specific inhibitor of MEK, inhibited bryo 1-induced ERK activity and DNA binding. Furthermore, PD98059 blocked the bryo 1-induced differentiation of Reh cells, as assessed by a number of features associated with lymphoid differentiation, including changes in morphology, cell growth arrest, attachment, and increased expression of the leukocyte integrin CD11c. Moreover, transient transfection of Reh cells with antisense MAP kinase oligonucleotides blocked bryo 1-induced expression of CD11c. Our analysis also shows that CD11c's gene promoter activity is augmented by bryo 1. Therefore, we conclude that activation of the MEK/ERK signaling pathway is necessary for bryo 1-induced differentiation of the pre-B Acute Lymphoblastic Leukemia cell line Reh.

Phase II study of bryostatin 1 in patients with relapsed multiple myeloma.

Bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan Bugula neritina, is a protein kinase C (PKC) modulator which has shown both preclinical and clinical activity in lymphoid malignancies. We conducted a phase II trial of bryostatin 1 administered at a dose of 120 microg/m2 by 72-h continuous infusion every 2 weeks in patients with relapsed multiple myeloma. Treatment was well tolerated with myalgias constituting the primaray toxicity. There were no responses in nine evaluable patients. The preclinical anti-lymphoid activity is strong enough to support further exploration of bryostatin 1 in different schedules and in combination therapy for multiple myeloma.

Chromosomal analyses of 52 cases of follicular lymphoma with t(14;18), including blastic/blastoid variant.

We have identified 52 patients of follicular lymphoma (FL) with t(14;18)(q32;q21). Histologically, the lymphomas were placed into six groups according to their cellular composition and growth pattern. Chromosome analysis revealed that all cases but one had additional secondary chromosomal abnormalities. The most frequent numerical aberrations were gains of chromosomes 7 (38%), X (36%), 5 (15%), 12 (15%), 18/der(18)t(14;18) (25%), and 21 (15%). Structural abnormalities of chromosome 1 were seen in 19 tumors (36%) affecting both arms with breakpoints clustered at 1p36. Other structural abnormalities included partial deletions of 6q, 10q, and 13q. Breakpoint at 8q24 was seen in four cases. The chromosome aberrations were correlated with the morphological subtypes of follicular lymphoma. Gain of chromosome 7 appeared to be associated with follicular large cell lymphoma. The incidence of trisomy 5 and 12, and 13q- was higher in follicular lymphoma with aggressive histological features than in low-grade lymphoma. In addition, complexity of the karyotype and high degree of polyploidy increased with the grade. The most valuable cytogenetic markers in the t(14;18) lymphomas are those involving 8q24 which was found exclusively in the blastic/blastoid variant FL. Therefore, chromosome analysis in relation to histologic pattern of follicular lymphoma can provide additional information in predicting tumor evolution and transformation to a higher-grade malignancy.

Evaluation of combretastatin A-4 prodrug in a non-Hodgkin's lymphoma xenograft model: preclinical efficacy.

Combretastatin A-4 prodrug (CA4P) is a new antitubulin agent currently in phase I/II clinical trials against solid tumors. We have previously reported on the in vitro activity of CA4P against a panel of malignant human B-lymphoid cell lines. In this study, we investigated the antitumor and the antiangiogenic activity of CA4P in our diffuse large cell lymphoma WSU-DLCL2-SCID mouse model. WSU-DLCL2 cells (10(7)) were injected s.c. into 5-week-old female ICR-SCID mice. Tumor-bearing mice were treated at the CA4P maximum tolerated dose (MTD) of 800 mg/kg in different dose/schedules. CA4P showed significant antitumor activity against this lymphoma model. Best results were seen when MTD was given in two and four divided doses (400 and 200 mg/kg, respectively). CA4P given in four divided doses (4 x 200 mg/kg) showed a log10 kill of 1.01, T/C of 11.7% and T-C of 12 days. Immunohistochemical staining using anti-CD31 antibody after 6, 24, 48 and 120 h treatment revealed a significant decrease in the number of tumor blood vessels after 24 h (about 80%). Only the periphery of treated tumors revealed the presence of blood vessels. Morphological examination of the tumors after tetrachrome staining showed a necrotic center in tumors of CA4P-treated animals. New blood vessel formation was noted to emerge in tumor tissues as early as 48 h following a single dose of CA4P. The G2/M arrest observed in vitro was not detected in vivo indicating predominance of the antiangiogenic effects with regard to antitumor efficacy in vivo. We conclude that CA4P has antiangiogenic activity in this lymphoma model and the use of this agent should be explored clinically in the treatment of non-Hodgkin's lymphoma.

Treatment-induced expression of anti-apoptotic proteins in WSU-CLL, a human chronic lymphocytic leukemia cell line.

Bryostatin 1 (bryo 1) has been shown to potentiate the anti-tumor activity of 2-chloro-2-deoxyadenosine (2-CdA) in chronic lymphocytic leukemia (CLL) and in the WSU-CLL cell line. However, like resistant CLL, WSU-CLL cells lose their sensitivity to bryo 1/2-CdA treatment. We report that 2-CdA-induced IAP expression may be a possible mechanism whereby resistance to apoptosis is acquired in these cells. In WSU-CLL cells, three members of the Inhibitors of Apoptosis (IAP) family were identified. Bryo 1 treatment of WSU-CLL cells leads to initiation of the apoptotic cascade and induced a marginal increase in XIAP protein expression. In contrast, 2-CdA treatment, alone or in combination with bryo 1, induced a substantial increase in survivin and XIAP proteins and phosphorylation of BAD. Bryo 1 alone induced caspase-7 and -9 dependent [poly ADP-ribose] polymerase (PARP) cleavage, while sequential treatment with bryo 1 (72 h) followed by 2-CdA (24 h) induced caspase-3,-7, and -9 dependent PARP cleavage and increased apoptosis. Although exposure to bryo 1 initiated apoptotic events, apoptosis was first enhanced by 2-CdA, and then reversed in a time-dependent manner by 2-CdA-induced expression of survival proteins. Taken together, resistance to bryo 1/2-CdA treatment may be the result of 2-CdA-induced IAP inhibition of the intrinsic apoptotic pathway caspases.

Effects of combretastatin A-4 prodrug against a panel of malignant human B-lymphoid cell lines.

Combretastatin A-4 (CA-4) is one of a family of compounds isolated from the South African willow tree Combretum caffrum. CA-4 was found to be active against murine melanoma and a variety of other human solid tumors. For the first time, we report the effect of CA-4 against a panel of malignant human B-lymphoid cell lines [early pre-B acute lymphoblastic leukemia (Reh), diffuse large cell lymphoma (WSU-DLCL2), chronic lymphocytic leukemia (WSU-CLL) and Waldenstrom's macroglobulinemia (WSU-WM)]. Our results indicate, using the prodrug form of CA-4, a concentration-dependent growth inhibition in all tested cell lines, although WSU-DLCL2 was more sensitive. Exposure to 4 nM CA-4 for 96 h induced 77% growth inhibition in Reh, 86% in WSU-CLL and 92% in WSU-WM. When used against the WSU-DLCL2 cell line, this same concentration of CA-4 was completely toxic. Morphological examination showed CA-4 induced the formation of giant, multinucleated cells, a phenomenon commonly found in mitotic catastrophe. Only minimal numbers of cells showing characteristics of apoptosis were detected. In WSU-DLCL2 cells, CA-4 (3 nM) induced the highest apoptosis (5%) after 48 h, while the percentage of dead cells was approximately 47%. Exposure of Reh, WSU-CLL, WSU-WM and WSU-DLCL2 cells for 24 h to 5 nM CA-4 induced 19, 28, 57 and 75% G2/M arrest, as determined by flow cytometry, respectively. Based on these preliminary studies, we believe that mitotic catastrophe is the predominant mechanism by which CA-4 induces cell death rather than apoptosis. Further studies to elucidate the mechanisms of CA-4 activity in vitro and in vivo are currently under investigation in our laboratory.

Non-Hodgkin's lymphoma: an analysis of the Metropolitan Detroit SEER database.

We evaluated incidence and survival trends of non-Hodgkin's lymphoma (NHL) in a large population-based cancer registry. Data regarding demographics, histology, incidence, and survival were obtained on all patients with NHL registered in the Metropolitan Detroit Cancer Surveillance System, a participant in the Surveillance Epidemiology and End Results (SEER) Program of the National Cancer Institute. Incidence and survival trends from 1973 through 1995 were evaluated and stratified based on age at diagnosis, sex, race, and tumor grade. There were 11,978 patients diagnosed with NHL and recorded in the Metropolitan Detroit SEER registry from 1973 to 1995. The age-adjusted incidence rate increased from 8.6 to 15.8 per 100,000, leading to an overall increase in incidence of 83% and an average annual increase of 3.2% per year. Incidence increased significantly (p < 0.05) over time in all age groups except the youngest (ages 0-19) and in all demographic groups studied. Incidence was highest in white men and lowest in black women. The incidence of both low-grade and intermediate/high-grade NHL increased significantly for each age group (p < 0.05) except the youngest (ages 0-19). In the oldest patients (70+ years), the incidence of intermediate/high-grade NHL was almost double that of low-grade NHL. Five-year relative survival increased from 64% (1973-1983) to 68% (1984-1991) for patients with low-grade NHL and from 40% to 44% for those with intermediate/high-grade NHL. The increase in relative survival was only seen in whites, however, with 5-year relative survival in blacks decreased from 53% (1973-1983) to 45% (1984-1991). In metropolitan Detroit, the current NHL epidemic affects all age groups except the very young (ages 0-19), both sexes, and both whites and blacks and is due to increases in the incidence of both low-grade and intermediate/high-grade NHL. Five-year survival rates have increased for whites but not for blacks.

Sequential treatment of a resistant chronic lymphocytic leukemia patient with bryostatin 1 followed by 2-chlorodeoxyadenosine: case report.

Bryostatin 1 (Bryo-1) has been shown to differentiate chronic lymphocytic leukemia (CLL) cells to the hairy cell leukemia phenotype. The purine analogue 2-chlorodeoxyadenosine (2-CdA) exhibits enhanced activity in patients with hairy cell leukemia compared to those with CLL. Here we present a case report of a patient diagnosed with resistant CLL and treated sequentially with Bryo-1 followed by 2-CdA for three cycles. Molecular and biochemical parameters relative to the sequential treatment with these agents in vivo were comparable to those found in the WSU-CLL cell line in vitro (R. M. Mohammad et al., Clin. Cancer Res., 4: 445-453, 1998; R. M. Mohammad et al., Biol. Chem., 379: 1253-1261, 1998). There was a significant reduction of lymphocyte count from 37.1 x 10(3)/microl before the treatment to 3.4 x 10(3)/microl after treatment, and partial remission was achieved 2 months after the treatment. The percentage of morphologically differentiated lymphocytes was increased from 3% before treatment to 92% with the first cycle of Bryo-1. Similarly, expression of CD22, a marker of differentiation, increased from 38% to 97% and was maintained at a high level for the duration of the treatment. Analysis of the molecular markers of apoptosis in isolated peripheral blood lymphocytes revealed an increase in the Bax:Bcl-2 ratio after treatment with Bryo-1 in cycles 2 and 3, with associated poly(ADP-ribose) polymerase cleavage after Bryo-1 and 2-CdA treatment. The deoxycytidine kinase: cytosolic 5'-nucleotidase activity ratio increased modestly after Bryo-1 treatment, indicating increased sensitivity of the peripheral blood lymphocytes to 2-CdA. In summary, we found that sequential treatment with Bryo-1 and 2-CdA caused a significant reduction in peripheral blood lymphocytes (CLL cells) with simultaneous induction of differentiation and the initiation of the Bax: Bcl-2 apoptotic pathway.

Bryostatin 1-induced modulation of nucleoside transporters and 2-chlorodeoxyadenosine influx in WSU-CLL cells.

WSU-CLL cells, a fludarabine resistant B-cell chronic lymphocytic leukemia cell line, has been shown to exhibit enhanced sensitivity to 2-chlorodeoxyadenosine (2-CdA) following 48-72 h exposure to bryostatin 1. For 2-CdA to manifest its chemotherapeutic activity, it must first enter the cell through one of several specific nucleoside transporter systems. We present data to show that bryostatin 1-induced enhanced influx of 2-CdA is in part the result of bryostatin 1-induced modulation of nucleoside transporters in WSU-CLL cells. The bi-directional equilibrative NBMPR sensitive transporters in WSU-CLL cells were significantly down-regulated 90 min post-exposure to 1-200 nM bryostatin 1. This down-regulation was evident up to 144 h. In contrast, WSU-CLL cells exhibited a transient increase in Na+-dependent concentrative 2-CdA influx from 48 to 96 h after bryostatin 1 exposure which was evident for a longer duration than that accounted for by the increase in deocycytidine kinase activity. These data may, in part, explain the enhanced efficacy of 2-CdA seen in WSU-CLL cells following 48-72 h exposure to bryostatin 1. It may raise questions as to the importance of the bi-directional transporters in determining the resistance or sensitivity of CLL cells to 2-CdA or other nucleoside analogues.

Phase II trial of bryostatin 1 in patients with relapsed low-grade non-Hodgkin's lymphoma and chronic lymphocytic leukemia.

Bryostatin 1 is a natural product isolated from the marine bryozoan Bugula neritina in 1982 and is currently undergoing evaluation in a number of malignancies. Twenty-five patients with relapsed, low-grade non-Hodgkin's lymphoma or chronic lyphocytic leukemia (CLL) received bryostatin 1 by 72-h continuous infusion every 2 weeks at a dose of 120 microg/m2 per course. Patients who progressed while receiving bryostatin 1 alone could participate in a feasibility study by receiving vincristine administered by bolus i.v. injection immediately after the completion of the bryostatin 1 infusion. The dose of vincristine was escalated in groups of three patients as follows: level 1, 0.5 mg/m2; level 2, 1.0 mg/m2; and level 3, 1.4 mg/m2 with vincristine doses capped at 2.0 mg for all patients. Bryostatin 1 alone resulted in one complete remission and two partial remissions. Nine patients received sequential treatment with bryostatin 1 and vincristine. The addition of vincristine at a dose of 2 mg was feasible and caused the expected dose-related sensory neuropathy. Phenotypic analysis by flow cytometric analysis on pre- and post-bryostatin 1-treated peripheral blood lymphocytes revealed up-regulation in the coexpression of CD11c/ CD22 on CD20+ B cells in two of four CLL patients studied, which is consistent with in vitro findings of differentiation of CLL cells to a hairy cell phenotype.

Bryostatin 1 induces ubiquitination and proteasome degradation of Bcl-2 in the human acute lymphoblastic leukemia cell line, Reh.

The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression.

The addition of bryostatin 1 to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy improves response in a CHOP-resistant human diffuse large cell lymphoma xenograft model.

The incidence of non-Hodgkin's lymphoma has been increasing at a rate of 4% per year since 1950; more than 62,000 cases will be diagnosed in the United States in 2000. Diffuse large cell lymphoma (DLCL) is the prototype of curable non-Hodgkin's lymphoma. Empirically designed chemotherapy regimens did not increase the cure rate of 30-40% achieved by the original four-drug regimen introduced in the 1970s [cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)]. We studied the antitumor effects of the CHOP regimen alone or in combination with a unique protein kinase C activator, bryostatin 1, on a xenograft model for resistant DLCL in mice with severe combined immune deficiency (WSU-DLCL2-SCID). In this model, the efficacy of bryostatin 1 given at 75 microg/kg, i.p., alone for 1 or 2 days [B(1x) and B(2x)]was compared with the efficacy of CHOP alone, bryostatin 1 + CHOP (B+CHOP) given concurrently, bryostatin 1 for 1 day followed by CHOP on day 2 [B(1x)-CHOP], and bryostatin 1 for 2 days followed by CHOP on day 3 [B(2x)-CHOP]. CHOP doses were as follows: (a) cyclophosphamide, 40 mg/kg, i.v.; (b) doxorubicin, 3.3 mg/kg, i.v.; (c) vincristine, 0.5 mg/kg, i.v.; and (d) prednisone, 0.2 mg/kg, every day for 5 days, p.o. Tumor growth inhibition (T/C), tumor growth delay (T-C), and log10 kill for B(1x), B(2x), CHOP, B+CHOP, B(1x)-CHOP and B(2x)-CHOP were 49%, 39%, 25.8%, 15.1%, 14.6%, and 12%; 6, 7, 16, 25, 12, and 15 days; and 0.6, 0.5, 2.2, 3.6, 1.7, and 2.0, respectively. To begin elucidating the mechanism whereby bryostatin 1 potentiated the effects of CHOP in the mouse model; we studied the effect of bryostatin 1 on Bax, Bcl-2, and poly(ADP-ribose) polymerase proteins in vitro and in vivo. Bax protein increased in a time-dependent manner without any measurable change in Bcl-2 expression. However, significant cleavage of the preapoptotic marker poly(ADP-ribose) polymerase was not recorded, and the percentage of apoptotic cells detected by flow cytometry increased only slightly (approximately 8%) after 96 h of bryostatin 1 exposure. The in vitro and in vivo results emphasize the superiority of combining bryostatin 1 with the CHOP regimen against the WSU-DLCL2 model. One possible mechanism may be the modulatory effects of bryostatin 1 on the Bax:Bcl-2 family of apoptosis-regulatory proteins. The use of this combination should be further explored clinically in the treatment of lymphoma.

Modulation of cIAP-1 by novel antitubulin agents when combined with bryostatin 1 results in increased apoptosis in the human early pre-B acute lymphoblastic leukemia cell line Reh.

Previous studies have shown that bryostatin 1 induces a decrease in the expression of the antiapoptotic protooncogene Bcl-2 in the human acute lymphoblastic leukemia (ALL) cell line Reh. This down-regulation has been shown to reduce drug resistance of the Reh cells to anti-tubulin polymerization agents. In the present study we investigated the effect of bryostatin 1 alone and in combination with novel anti-tubulin agents (dolastatin 10 and auristatin PE) and the chemotherapeutic vincristine on the inhibitor of apoptosis protein cIAP-1. Cells were cultured with bryostatin 1 (1 nM), dolastatin 10 (0.1 ng/ml), auristatin PE (0.1 ng/ml), or vincristine (0.5 ng/ml) alone or the combination of these anti-tubulins with bryostatin 1. Western blots were conducted to assess the effects of the above agents on cIAP-1 protein level. Flow-cytometric analysis [7-amino-actinomycin D (7AAD)] was conducted to assess apoptosis as well as staining for morphology using tetrachrome stain. Our results show that cIAP-1 is induced in a time-dependent fashion after bryostatin 1 exposure up to 72 h. However, upon treatment of cells with a combination of bryostatin 1 and dolastatin 10 or auristatin PE, the induction of cIAP-1 was abolished, leading to a significant increase in apoptosis. The initial 24- and 48-h reduction in cIAP-1 protein level recorded in the bryostatin 1 and vincristine combination recovered to control levels by 72 h. We believe that this phenomenon is responsible for the reduced apoptosis recorded in this combination. Results of this study should prove useful in guiding the clinical application of these novel agents in the treatment of ALL.

Clonal preservation of human pancreatic cell line derived from primary pancreatic adenocarcinoma.

Adenocarcinoma of the pancreas generally remains an incurable disease by available treatment modalities, demanding the development of a suitable cell-culture/animal model and the discovery and evaluation of novel therapeutic agents. We report the clonal preservation of a human pancreatic cell line (KCI-MOH1) established from a 74-year-old African-American man diagnosed with pancreatic cancer. Initially the human primary tumor was grown as a xenograft in SCID mice and, subsequently, a cell line was established from tumors grown as a xenograft as reported in our earlier publication. The molecular characterization of the primary tumor, the tumors grown as xenograft, and the cell line all revealed similar genotypic properties. By using an automated DNA sequencer, a K-ras mutation (codon 12, GGT to CGT, Gly to Arg) was detected in the pancreatic tumor tissue taken from the patient, whereas no p53 mutation was detected. The same K-ras mutation and unaltered p53 was also found in the xenograft tumor and in the KCI-MOH1 cell line. Chromosome analysis of the cultured cells revealed: 42,XY,add(3)(p11.2),der(7)t(7;12) (p22;q12),-10,-12,add (14)(p11),-18,add (20)(q13),-22/84, idemx2, which is the same chromosome complement found in xenograft tumors. The KCI-MOH1 cell line grows well in tissue culture and forms tumors in the SCID mice when implanted subcutaneously, as well as in orthotopic sites. The KCI-MOH1 cell line-derived SCID mouse xenograft model was used for efficacy evaluation of bryostatin 1, auristatin-PE, spongistatin 1, and gemcitabine alone and in combination. Tumor growth inhibition (T/C expressed as percentage), tumor growth delay (T - C), and log 10 kill for these agents were 38%, 22 days, and 0.53; 15%, 30 days, and 0.80; 24%, 25 days, and 0.66; and 10%, 33 days, and 0.90, respectively. When given in combination, two of seven gemcitabine + auristatin-PE-treated animals were free of tumors for 150 days and were considered cured. Animals treated with a combination of bryostatin 1 and gemcitabine and a combination of spongistatin and gemcitabine produced remissions in only one of seven mice. From these results, we conclude that (a) this is the first study illustrating that clonal characteristics of primary pancreatic tumors remained unchanged when implanted in mice and as a permanent cell line grown in vitro; and (b) there is a synergistic effect between gemcitabine and selected marine products tested in this study, which is more apparent in the gemcitabine and auristatin-PE combination. The results of this preliminary study suggest that these agents should be explored clinically in the treatment of pancreatic cancer.

Bax:Bcl-2 ratio modulation by bryostatin 1 and novel antitubulin agents is important for susceptibility to drug induced apoptosis in the human early pre-B acute lymphoblastic leukemia cell line, Reh.

The ratio of Bax to Bcl-2 protein can determine whether cells will die via apoptosis or be protected from it. Reh was found to express a high basal level of Bcl-2 but was lacking of Bax protein expression. Treatment with bryostatin 1 induced a down-regulation in Bcl-2 protein that was not accompanied by an obvious Bax protein induction or apoptosis. These results suggest that a decreased level of Bcl-2 alone in this cell line is not sufficient for apoptosis induction. In an effort to identify the mechanism whereby apoptosis could be induced in this ALL model, we treated Reh cells with three microtubule inhibitors: dolastatin 10, auristatin PE and vincristine, in the presence and absence of bryostatin 1. When used alone, only dolastatin 10 induced apoptosis that was detected morphologically, and by flow cytometry. Western blots revealed that dolastatin 10-induced apoptosis was accompanied by the induction of Bax protein and the reduction in Bcl-2 protein. Auristatin PE and vincristine induced both Bax and Bcl-2 protein, leaving the Bax:Bcl-2 ratio constant. Reh cells pretreated for 24 h with bryostatin 1 followed by dolastatin 10, auristatin PE or vincristine showed significant apoptosis which was accompanied by Bcl-2 protein down regulation and Bax protein up regulation. We conclude that: (1) expression of bax is necessary for apoptosis-induction in this model; (2) a decrease in Bcl-2 level alone is not sufficient and might not be necessary for apoptosis-induction; and (3) the ratio of Bax:Bcl-2 plays a critical role in susceptibility to apoptosis in Reh cells. The results from this study should prove useful in guiding the clinical application of these novel agents in the treatment of acute lymphoblastic leukemia.

Neutron and photon clonogenic survival curves of two chemotherapy resistant human intermediate-grade non-Hodgkin lymphoma cell lines.

BACKGROUND: The potential role of neutron therapy in the management of intermediate-grade non-Hodgkin lymphoma (IGNHL) has not been examined because of the belief that the anticipated radiobiological effectiveness (RBE) would be uniformly very low. PURPOSE: To determine the fast neutron RBE for two chemotherapy-resistant IGNHL cell lines. METHODS AND MATERIALS: Conventional soft agar clonogenic survival curves following irradiation by 60Co and fast neutron were established for two IGNHL cell lines. These cell lines, WSU-DLCL2 and SK-DHL2B, were found in previous studies to be able to repair sublethal damage, and were also resistant to L-Pam and doxorubicin chemotherapy. RESULTS: When the surviving fraction after 2 Gy photon was chosen as the biological endpoint, the RBE for WSU-DLCL2 and SK-DHL2B measured 3.34 and 3.06. Similarly, when 10% survival was considered, the RBE for these two cell lines measured 2.54 and 2.59. The RBE, as measured by the ratios alpha neutron/alpha photon, for WSU-DLCL2, SK-DHL2B cell lines are 6.67 and 5.65, respectively. These results indicate that the RBE for these IGNHL cell lines is higher than the average RBE for cell lines of other histological types. CONCLUSION: Fast neutron irradiation may be of potential value in treating selected cases of IGNHL.

Molecular effects of taxol and caffeine on pancreatic cancer cells.

Pancreatic cancer is the fifth leading cause of cancer related deaths in the United States. Despite many recent advances in the treatment modalities, the mortality rate still remains very high. Paclitaxel (Taxol) and Caffeine have been used for the treatment of this disease, however the molecular mechanisms of these agents are not fully understood, which may be partly responsible for the failure of these agents in the treatment of pancreatic cancer. Human pancreatic adenocarcinoma cell lines, HPAC and PANC-1 containing wild-type and mutant p53 respectively, were used to investigate the effects of Taxol and Caffeine on cell growth, and their effects on the modulation of cell cycle and apoptosis related genes. Protein extracts from these cells treated with 100 nM of Taxol or 4 mM of Caffeine were subjected to Western blot analysis for this study. Drug treated cells were also analyzed to calculate the number of cells undergoing apoptosis. Dose and time dependent growth inhibition was observed in both PANC-1 and HPAC cells when treated with either Taxol or Caffeine. Western blot analysis showed an up-regulation of p21WAF1 in both cell lines treated with either Taxol or Caffeine. Furthermore, down-regulation of cyclin B and cdk1 was observed in Taxol and Caffeine treated HPAC cells. However, the results were drastically different in PANC-1 cells where cyclin B was down regulated only by Caffeine treatment and the level of cdk1 protein was undetectable in this cell line. Moreover, up-regulation of p53 and down-regulation of Bcl-2 was observed only in HPAC cells treated with Taxol. Apoptotic cell death analysis showed increasing number of cells undergoing apoptosis between 24 and 48 h of Caffeine treatment, however only Taxol showed greater than 50% cells under-going apoptosis only in HPAC cells. The up-regulation of p21WAF1 and down-regulation of cyclin B and cdk1 suggest their possible roles in G2/M cell cycle arrest caused by both Taxol and Caffeine as reported earlier. From these results we conclude that the differential molecular changes observed in this study may determine the cellular effects of these agents on pancreatic adenocarcinoma cells and that the effects of chemotherapeutic agents may be determined by the endogenous status of p53 mutation and, in turn, may determine the therapeutic effects of these agents in the treatment of pancreatic cancer.

Infrared spectroscopic study of bryostatin 1-induced membrane alterations in a B-CLL cell line.

Previous studies on intact cells have shown that bryostatin 1 (Bryo 1) induces significant alterations in the membranes of WSU-CLL cells (a drug-resistant B-CLL cell line), changes which may play an important role in the mechanism of reduced drug resistance of B-CLL cells to 2-chlorodeoxyadenosine (2-CdA). However, it is not clear whether the plasma membranes or the mitochondria, or both are involved; nor is it known which of these two targets is more important for regaining the cells former drug sensitivity. For the present study, we treated WSU-CLL cells with Bryo 1, isolated plasma membranes and mitochondria, and then subjected the purified fractions to infrared (IR) spectroscopic and chromatographic analyses. IR spectroscopy revealed a decreased glycosylation of both plasma membranes and mitochondria in Bryo 1-treated cells compared to untreated cells. The amount of lipid relative to protein was increased in both types of membranes, but considerably more enhanced in the plasma membrane fraction of the Bryo 1-treated cells than in mitochondria. Quantitative lipid analysis by thin layer chromatography also revealed that Bryo 1 treatment significantly increased the phospholipid content in plasma membranes, whereas the lipids in the mitochondria remained essentially unchanged. Changes in lipid composition were quite dramatic for plasma membranes where phosphatidylcholines were decreased by 50%, phosphatidylethanolamines doubled and sphingomyelins increased five-fold compared to the lipid composition in plasma membranes of untreated cells. In addition, the IR spectroscopic analysis provided evidence for an increased plasma membrane fluidity in Bryo 1-treated cells, whereas the fluidity of the mitochondria remained essentially unchanged; marker bands indicating mitochondrial DNA decreased upon Bryo 1 treatment. These results suggest that Bryo 1 increases the sensitivity of WSU-CLL cells to chemotherapeutic agents such as 2-CdA by action on two cell targets: (1) introduction of significant changes in plasma membrane permeability or fluidity through modifications in lipid content and composition as well as by reducing the surface glycosylation; (2) introduction of changes in lipid and DNA content of the mitochondria. Small alterations in the lipid composition of the mitochondria may provide the conditions for an altered proton gradient and transmembrane potential leading to apoptosis and decreased cell survival.

Radiobiological characterization of two human chemotherapy-resistant intermediate grade non-Hodgkin's lymphoma cell lines.

Intermediate grade non-Hodgkin's lymphoma (IGNHL) is generally considered a radiosensitive tumor that can be controlled with moderate radiation doses. Cell-survival curves of cell lines derived from IGNHL have been typically described to exhibit small or no shoulder, implying inability to accumulate or repair sublethal radiation damage. We characterize in this report the clonogenic radiation survival curves of two human IGNHL cell lines, WSU-DLCL2 and SK-DHL2B, established from patients who expired after having exhibited chemotherapy resistance of their tumors. The cells were irradiated with 60Co radiation at a dose rate of 85-100 cGy/min and cell survival data were analyzed according to the linear quadratic model. The alpha/beta values for WSU-DLCL2 and SK-DHL2B cells are 2 and 8.6, respectively. The corresponding SF2 are 0.42 and 0.35, respectively. Both cell lines are able to repair radiation-induced sublethal damage. These data indicate that these cells are only moderately radiosensitive.