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Gastrointestinal Oncology Research Laboratory, Division of Solid Tumor Oncology, Department of Medicine [M. M., C. R., G. K. S.] and Program of Molecular Pharmacology and Experimental Therapeutics [F. S., Y. S.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021

² To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-8324; Fax: (212) 717-3320; E-mail: schwartg{at}mskcc.org

	Abstract

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Gastric cancer is one of the leading causes of cancer death throughout the world. It is a disease in desperate need of new therapeutic approaches. Docetaxel, a semisynthetic taxane, has shown potent activity against a broad range of solid tumors. However, in gastric cancer, response rates to docetaxel remain only 20%. In these studies we show that flavopiridol, a cyclin-dependent kinase inhibitor, potentiates docetaxel-induced apoptosis 3-fold in MKN-74 human gastric cells. This effect is sequence dependent, such that flavopiridol must follow docetaxel to induce this effect. Docetaxel induces transient arrest in the M phase of the cell cycle. Cells exit mitosis in a specific time window without cytokinesis with a decrease in cyclin B1/cdc-2 kinase activity and MPM-2 labeling. Flavopiridol treatment of docetaxel-treated cells enhances the exit from mitosis with a more rapid decrease in mitotic markers including MPM-2 labeling and cyclin B1/cdc2 kinase activity. In contrast, pretreatment with flavopiridol prevents cells from entering mitosis by inhibiting cyclin B1/cdc-2 kinase activity, thus antagonizing the docetaxel effect. The testing of this combination against MKN-74 xenografts confirms the sequence dependency. Treatment of MKN-74 tumor-bearing xenografts with docetaxel at a dose of 10 mg/kg followed 3–7 h later by flavopiridol at a dose of 2.5 mg/kg resulted in a 1–18% decrease in tumor volume. In contrast, treatment with docetaxel alone at this same dose resulted in a 394% increase in tumor volume. When flavopiridol was given immediately after docetaxel, the effect was not statistically different from that of docetaxel alone. The reverse combination of flavopiridol followed 7 h later by docetaxel was similar to treatment with docetaxel alone. Flavopiridol alone had no effect in this tumor model. Thus, flavopiridol, when combined with docetaxel in a sequence-specific manner, may provide a completely new therapeutic approach in the treatment of gastric cancer.

	Introduction

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Gastric cancer is a leading cause of cancer death throughout the world. In the United States, there will be 23,000 cases of gastric cancer diagnosed this year, and this will be associated with 12,800 deaths (1). These patients invariably die of metastatic disease. Recent randomized studies using 5-fluorouracil-based chemotherapies indicate response rates of 10–20%, with a median survival of 7–8 months (2). In recent years the taxanes, docetaxel (Taxotere) and paclitaxel (Taxol), have been introduced into the treatment of solid tumor cancers. This has resulted in some success, especially in the treatment of metastatic breast cancer, in which response rates of up to 68% in untreated and 57% in pretreated patients have been reported (3). However, despite the highly improved response rates in advanced metastatic breast disease, success in patients with metastatic gastric cancer has been less compelling. The results of the Phase II Eastern Organization for Research and Training Early Clinical Trials Group indicate a response rate to single-agent docetaxel of 24%, with only partial responses and a median survival of 7.5 months (range, 3–11+ months; Ref. 4). These results are comparable with the randomized studies reported with combinations of 5-fluorouracil and cisplatin (2). Thus, new therapies are desperately needed in the treatment of gastric cancer. An advance in this area would have a major impact on the outcome of a large number of patients with this disease.

Further opportunities to enhance the effectiveness of the taxanes are being investigated. One approach is to add classic cytotoxic agents to the taxanes in the hope that this will increase the response rate and result in an overall improvement in survival. However, such standard combination therapies will undoubtedly be associated with increased toxicities that may outweigh any potential benefit of the drug combination. Another approach is to combine the taxanes with cell cycle inhibitors that could potentiate taxane-induced apoptosis and result in an increased antitumor effect. One promising candidate along this line is flavopiridol, a synthetic flavone currently undergoing Phase I and II clinical trials (5). Flavopiridol has been shown in vitro to inhibit tumor cell growth at nanomolar concentrations through blockade of cell cycle progression at G₁ or G₂ (6). It is a potent inhibitor of CDKs³ with respect to the ATP binding site. Inhibition of CDKs including CDK-1 (cdc-2), CDK-2, CDK-4, and CDK-7 and hypophosphorylation of pRb have also been reported (7, 8). We have reported previously that flavopiridol at nanomolar concentrations significantly enhances the induction of apoptosis by mitomycin-C and paclitaxel in gastric and breast cancer and by CPT-11 in colon cancer cell lines (9–11). Synergism between flavopiridol and paclitaxel has also been observed against A549 non-small cell lung cancer cells (12). These studies indicate that the combination of paclitaxel and flavopiridol is highly sequence dependent, such that paclitaxel should precede flavopiridol to achieve the maximal effect (10, 12).

Taxanes promote microtubular aggregation and block cells in metaphase (13). The importance of mitotic block in induction of apoptosis in response to the taxanes has been shown by various groups using antisense of cyclin B1 to abrogate cdc-2 kinase activity (14). The prevention of mitotic block also prevents cell death. However, the mechanism defining mitotic block to induce apoptosis by docetaxel is not clearly understood. Docetaxel has shown superior cytotoxic potency to paclitaxel against murine and human cell lines, which may be due to a higher affinity for microtubules (15). Both taxanes also trigger apoptosis in cancer cells via bcl-2 phosphorylation and inactivation (16). However, docetaxel has been shown to be 100-fold more potent than paclitaxel in terms of the ability to phosphorylate the antiapoptotic target bcl-2 (16). Therefore, docetaxel may have theoretical advantages over paclitaxel in terms of its clinical development. Randomized trials comparing docetaxel with paclitaxel are under way.

We elected to examine the effect of flavopiridol on docetaxel-treated MKN-74 gastric cancer cells. In this study we have characterized the apoptotic and cell cycle events associated with this combination therapy. Our results indicate that flavopiridol potentiates the effect of docetaxel both in vitro and in vivo. This effect is associated with the induction of apoptosis when flavopiridol is administered after docetaxel. Pretreatment of cells with flavopiridol inactivates the cyclin B1/cdc-2 kinase. This prevents the mitotic arrest by docetaxel from occurring in the context of a properly activated cdc-2 kinase, thus rendering the sequence of flavopiridol followed by docetaxel combination inactive. The importance of sequence in flavopiridol’s potentiation of docetaxel was confirmed in mice bearing MKN-74 xenografts.

	Materials and Methods

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Cell Culture.
The human gastric cancer cell line MKN-74 was graciously supplied by Dr. E. Tahara (Hiroshima University, Hiroshima, Japan). Cells were maintained in Eagle’s minimal essential media supplemented with 10% heat-inactivated fetal bovine serum (HyClone), penicillin, and streptomycin at 37°C in 5% carbon dioxide. Cells were tested as Mycoplasma free.

QFM.
The nuclear morphology of the cell (multinucleated and apoptotic) was determined by staining nuclear chromatin with 4',6-diamidino-2-phenylindole (Sigma, St. Louis, MO). Apoptotic cells have condensed and fragmented chromatin. The percentage of apoptosis was determined by counting the cells and scoring them for the incidence of apoptosis using an Olympus BH2-DM2U2UV Dichetomic Mirror cube filter (Olympus, Lake Success, NY). The protocol has been described previously (10). Briefly, MKN-74 cells were cultured for 24–48 h (approximately 60% confluent) and treated according to one of the following conditions: (a) drug-free media; (b) 300 nM flavopiridol (graciously supplied by Dr. Edward Sausville, National Cancer Institute, Bethesda, MD) alone for 24 h; (c) 100 nM docetaxel (Taxotere; Aventis Pharmaceuticals, Bridgewater, NJ) alone for 18 h; (d) 300 nM flavopiridol and 100 nM docetaxel together for 18 h; (e) 300 nM flavopiridol for 24 h followed by removal of media containing flavopiridol and addition of media with 100 nM docetaxel for 18 h; (f) the same drugs given in reverse order; and (g) 100 nM docetaxel for 18 h followed by removal of drug media and addition of fresh media without drug. In sequential therapy, the floating cells were collected and added back for subsequent treatment. At the end of treatment, adherent cells were trypsinized, pooled with floating cells, washed with PBS, and fixed in 3% paraformaldehyde for 10 min at room temperature. Cells were stained with 4',6-diamidino-2-phenylindole for 30 min at room temperature in the dark. The aliquots of cells were taken to prepare slides, and duplicate samples of 400 cells each were counted and scored for the incidence of apoptotic chromatin condensation.

MPM-2/Propidium Iodide Bivariate Flow Cytometry.
The MKN-74 cells were treated as described above and fixed and labeled with MPM-2 antibody as described previously (10). The MPM-2-positive (mitotic) cells show increased green fluorescence; thus shift above the baseline of the dot plot. Mitotic index was defined as percentage of MPM-2-positive cells.

Cyclin B1/cdc-2 Kinase Activity Assay.
The MKN-74 cells were treated with docetaxel and flavopiridol as discussed above. The cell lysates were prepared as described previously (10). Soluble protein (200 µg) was incubated with 1 µg of anti-cyclin B1 antibody (catalogue number sc-245; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 2 h. Immune complexes were then precipitated with 40 µl of immobilized rProtein A (RepliGen) overnight at 4°C, and kinase activity was assayed as described previously.

Immunoblot Analysis.
Protein lysates prepared for kinase assays were used for immunoblotting. Western blots were prepared as described previously. The membranes were probed with mouse monoclonal antibodies specific to ppRb (PharMingen) and mouse monoclonal cyclin B1 (catalogue number sc-245; Santa Cruz Biotechnology). The membranes were treated with a secondary sheep antimouse horseradish peroxidase or donkey antirabbit horseradish peroxidase antibody for 1 h at room temperature. Detection was done by enhanced chemiluminescence reagents (Dupont NEN Life Science Products, Boston, MA) according to the manufacturer’s protocol. The levels of expression were quantified using a densitometric scanning system.

Xenograft Growth Assay.
The general procedure used in the experiments has been described previously (17). Athymic-NCr-nu male mice (8–10 weeks old) were inoculated s.c. in flank with MKN-74 cells mixed with Matrigel (Becton Dickinson). Treatment was started on the third day after the inoculation of tumor, and animals were treated with the maximum tolerated dose of docetaxel either as single agent or in combination. The maximum tolerated dose of flavopiridol was used as a single agent, and the dose was decreased in combination therapy to minimize cytotoxicity. The average tumor volume at the day of treatment was 27–29 mm³. Mice received docetaxel alone (10 mg/kg), flavopiridol alone (10 mg/kg), or docetaxel (10 mg/kg) followed by flavopiridol (2.5 mg/kg) 0, 3, 7, or 16 h later. One group was treated in the reverse order, such that flavopiridol was administered first, and docetaxel was administered 7 h later. Mice in the control group were given vehicle (PBS) alone. All drugs were administered i.p. twice a week, for a total of four injections. Tumors were measured every 3–4 days with calipers, and tumor volumes were calculated by the formula 4/3 x x r³ [r = (larger diameter + smaller diameter)/4]. The percentage of tumor regression was calculated as the percentage ratio of difference between baseline and final tumor volume to the baseline volume. These studies were performed in accordance with the Principles of Laboratory Animal Care (NIH Publication No. 85–23, released 1985).

Biostatistical Analysis.
All experiments were done in duplicate and repeated at least three times unless otherwise indicated. The statistical significance of the experimental results was determined by the two-sided t test.

	Results

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Flavopiridol Enhances Docetaxel-induced Apoptosis in a Sequence-dependent Manner.
Human gastric cancer MKN-74 cells were treated with 100 nM docetaxel (a clinically achievable concentration) and 300 nM flavopiridol (a concentration established to inhibit CDKs) individually, concurrently, and sequentially as docetaxel for 18 h followed by flavopiridol for 24 h, or the same drugs given in reverse sequence. The treatment times for each drug were chosen based on our previous studies with paclitaxel and flavopiridol (10). As determined by QFM, under these treatment conditions, docetaxel for 18 h or flavopiridol for 24 h (Doc₁₈ or F₂₄) induced apoptosis in 2% of the cells. The sequential treatment of MKN-74 cells with docetaxel for 18 h followed by flavopiridol for 24 h (Doc₁₈F₂₄) induced apoptosis in 30 ± 2% of the cells (Fig. 1), which was significantly greater than that observed for docetaxel followed by drug-free media (11 ± 2%; Doc₁₈ND₂₄; P < 0.005). Treatment of Doc₁₈F₂₄ cells for an additional 24 h in drug-free media (Doc₁₈F₂₄ND₂₄) did not further enhance the induction of apoptosis (25 ± 2%) when compared with Doc₁₈F₂₄ (30 ± 2%). Under these treatment conditions (Doc₁₈ND₂₄, Doc₁₈F₂₄, and Doc₁₈F₂₄ND₂₄), an examination of cells by fluorescence microscopy indicated that these cells had acquired a multinucleated phenotype, a characteristic that has been attributed to aberrant mitosis following taxane therapy (18). When the cells were treated in the reverse order such that flavopiridol treatment preceded docetaxel (F₂₄Doc₁₈), only 2 ± 1% of the cells underwent apoptosis. This was no different than the induction of apoptosis by flavopiridol for 24 h followed by drug-free media (F₂₄ND₂₄; 2 ± 1%). Incubation of cells treated first with the reverse order of flavopiridol followed by docetaxel and then with an additional 24 h in drug-free media (F₂₄Doc₁₈ND₂₄) resulted in apoptosis in only 4 ± 1% of the cells. This was significantly less than the 11 ± 2% observed with Doc₁₈ND₂₄ (P < 0.005), indicating an antagonism to the induction of apoptosis when flavopiridol preceded docetaxel.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Flavopiridol potentiates docetaxel-induced apoptosis in MKN-74 cells. Induction of apoptosis in MKN-74 cells with docetaxel (100 nM) alone, flavopiridol (300 nM) alone, in combination or in sequential treatment of docetaxel followed by flavopiridol, or the same drugs given in reverse sequence. Cells were counted and scored for apoptotic chromatin condensation by the QFM method described previously (1) and in "Materials and Methods." Bars represent the mean (±SD) number of cells counted that undergo apoptosis as a percentage of 400 or more total cells randomly counted in duplicate samples.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Cyclin B1/cdc-2 kinase activity is transiently up-regulated after docetaxel treatment, and concurrent treatment or pretreatment with flavopiridol inhibits kinase activation by docetaxel. MKN-74 cells were treated with docetaxel and flavopiridol, and cell extracts were prepared. Two hundred µg of protein from these cells were immunoprecipitated with anti-cyclin B1, and the precipitates were assayed for kinase activity using histone H1 as substrate as described in "Materials and Methods." The experiment was repeated a minimum of three times, and representative data of one experiment are presented.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Cyclin B1 is up-regulated after docetaxel treatment. Lysates were prepared from MKN-74 cells treated under various conditions as described in "Materials and Methods." The Western blots were probed for anti-cyclin B1. The equal loading of protein was examined initially by amido black and later by measuring tubulin expression.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. pRb is hypophosphorylated after docetaxel removal. Lysates were prepared from MKN-74 cells treated under various conditions as described in "Materials and Methods." The Western blots were probed for anti-ppRb. The equal loading of protein was examined initially by amido black and later by measuring tubulin expression.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. The effect of docetaxel and flavopiridol on growth of established MKN-74 xenografts in nude mice. Mice were treated with docetaxel (Doc) and flavopiridol (flavo) alone or in combination as docetaxel with flavopiridol (Doc+F) or docetaxel followed by flavopiridol in 3 (DocF3), 7 (DocF7), and 16 (DocF16) h or flavopiridol followed by docetaxel after 7 h (FDoc7) on days 3, 6, 10, and 13 (arrows) according to the protocol described in "Materials and Methods." Tumor volumes were measured as described in "Materials and Methods," and mean log change in volume (calculated as volume/baseline volume) was plotted against time (days).

Mice that were treated with only docetaxel lost an average of 6 ± 9% body weight, whereas mice treated with docetaxel followed 3 or 7 h later by flavopiridol lost an average of 10 ± 6% body weight, indicating that the time intervals between therapies did not render the combinations more toxic. This would indicate that a difference in weight loss between these treatment conditions does not explain the significant difference in response. No animal deaths were observed with docetaxel alone or with docetaxel followed 0, 3, 7, or 16 h later by flavopiridol.

	Discussion

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We have previously reported that flavopiridol enhances the induction of paclitaxel-induced apoptosis in vitro in both breast and gastric cancer cells and that this effect was sequence dependent, such that paclitaxel therapy needed to precede the flavopiridol treatment (10). In this study we elected to determine whether these observations could be extended to another taxane, docetaxel, both in vitro and in vivo. Our studies indicate that the flavopiridol enhanced docetaxel-induced apoptosis by 2–3-fold in MKN-74 gastric cancer cells. This effect was highly sequence dependent, such that docetaxel needed to precede flavopiridol treatment to achieve this effect. In addition, pretreatment with flavopiridol before docetaxel antagonized the docetaxel effect. The downstream events that execute the docetaxel-induced cell death involve activation of caspases. In combination therapy of docetaxel followed by flavopiridol, there was enhancement of caspase-3 activation and poly(ADP-ribose) polymerase cleavage (data not shown).

We have extended these observations to an identical in vivo model. Using MKN-74 xenografts, our results again indicate that flavopiridol enhanced the effect of docetaxel. This effect was also highly sequence dependent. In fact, when flavopiridol was administered concomitantly with docetaxel or in the reverse order, the changes in tumor regression were not significantly different than those seen with docetaxel alone. It was only when flavopiridol followed docetaxel that a decrease in tumor volume relative to docetaxel was noted. This effect was both sequence and time dependent, such that the maximum effect was observed when flavopiridol followed docetaxel by 3 or 7 h. However, even with a 16-h interval, the effect of the combination was still greater than that seen with docetaxel alone.

The results presented here indicate that docetaxel (Doc₁₈) as a single agent induces mitotic arrest (i.e., increase in expression of mitotic markers such as MPM-2 labeling, cyclin B1 expression, and cyclin B1/cdc-2 kinase activity) and increased phosphorylation of pRb. We do not observe significant apoptosis at this time. Flavopiridol on untreated cells directly inhibits CDK2 and CDK4, the two kinases critical for pRb phosphorylation. This accounts for the marked decrease in pRb phosphorylation and the decrease in the S-phase population observed on the MKN-74 cells treated with flavopiridol alone (F₂₄). In contrast, when cells arrested in mitosis by docetaxel exit the M phase and enter G₁ (Doc₁₈ND₂₄), there is a decrease in mitotic markers and a decrease in cyclin B1/cdc-2 kinase activity, and the cells do not undergo cytokinesis (i.e., cells remain with 4n DNA content). We refer to this stage as pseudo-G₁ (10). pRb is found in the dephosphorylated form in the preceding hours of G₁ and needs to undergo phosphorylation to initiate the G₁-to-S-phase transition (22). Thus, pRb dephosphorylation is a marker of G₁ entry. This explains the decrease in pRb phosphorylation observed with Doc₁₈ND₂₄, as compared with Doc₁₈ alone. We also observed a significant increase in apoptosis during Doc₁₈ND₂₄ compared with Doc₁₈ alone. This may be due to abnormal exit of cells from mitosis.

However, with the addition of flavopiridol on docetaxel-treated cells (Doc₁₈F₂₄), there is a greater decrease in both cyclin B1/cdc-2 kinase activity and MPM-2 labeling (22% versus 11%) when compared with docetaxel followed by drug-free medium (Doc₁₈ND₂₄). This results in an increased accumulation of hypophosphorylated pRb, indicating greater mitotic exit and entry into pseudo-G₁. This greater degree of inhibition could not be explained by the decrease in cyclin B1 protein expression, which, in fact, was greater for Doc₁₈ND₂₄ than for Doc₁₈F₂₄. Instead, this must be due to direct inhibition of cyclin B1/cdc-2 kinase by flavopiridol. Collectively, these results indicate that flavopiridol after docetaxel treatment enhances the exit of cells out of mitosis with more rapid entry into a pseudo-G₁ state.

With the reverse combination of flavopiridol followed by docetaxel (F₂₄Doc₁₈), there was no increase in cyclin B1 expression or in the activation of cyclin B1/cdc-2 kinase when compared with docetaxel alone (Doc₁₈). This resulted in a lower level of phosphorylated pRb. This is consistent with the arrest in G₁ (4% in G₁ with Doc₁₈ alone versus 50% with F₂₄Doc₁₈) and a decrease in both the 4n (G₂ + M) and M-phase population (70% and 62%, respectively with Doc₁₈ alone to 43% and 22% with F₂₄Doc₁₈). Docetaxel-induced apoptosis is dependent on activation of cyclin B1/cdc-2 kinase and mitotic block. It has been shown that the prevention of mitotic block during paclitaxel treatment by use of an antisense to cyclin B1 also prevents cell death (14). Thus, pretreatment with flavopiridol antagonized the effect of docetaxel on these cells. Furthermore, the inhibition of docetaxel-induced cytotoxicity by flavopiridol (by cdc-2 kinase inactivation) confirms the importance of mitotic block and exit in docetaxel-induced apoptosis and provides an explanation for the ineffectiveness of the reverse combination (F₂₄Doc₁₈). Despite this antagonism in vitro, we did not observe this type of antagonism in vivo when flavopiridol was administered 7 h before docetaxel. This may simply reflect differences observed when tumor cells are exposed to drug in monolayers as opposed to tumor xenografts. Alternatively, in vivo we may have missed the time window in which antagonism of flavopiridol could have been observed (for example, flavopiridol followed by docetaxel in 3 h rather than 7 h). However, this was not tested in this tumor model because our goal was to maximize the effect of the drug combination.

A Phase I study of weekly, sequential docetaxel followed by flavopiridol therapy is now under way at our cancer center. From the animal studies, it would appear that both the sequence of drug administration and the time between drug administrations are crucial in order for flavopiridol to augment the effect of docetaxel. It is clear from the studies that giving the two drugs at comparable times will lead to effects that are no greater than docetaxel alone. It seems from the animal data that an interval of 3–7 h between sequential docetaxel and flavopiridol is needed to produce this effect. Interestingly, in our evaluation of sequential irinotecan and flavopiridol, the maximum enhancement was again observed with a 7-h interval between the initial administration of irinotecan and the subsequent treatment with flavopiridol (11). Admittedly, it is difficult to extrapolate animal data to the development of human clinical trials. Although there may be some specific metabolic effect in the mice that necessitates these drug intervals, the data do suggest a unique biological window in which the application of flavopiridol relative to the chemotherapy is critical to achieve a chemopotentiating effect. This may be based on the specific cell cycle effects of each drug under investigation. We do not believe this was due to the i.p. administration of flavopiridol because the drug is rapidly absorbed when given by i.p. administration in vivo. Therefore, in our Phase I study, we plan to use this preclinical data, such that patients with advanced gastrointestinal cancers will be treated with a fixed dose of weekly docetaxel followed in 4 h by escalating doses of flavopiridol administered over 1 h. We hope that this will develop into a new approach in the treatment of gastric cancer, which is in desperate need of new therapeutic approaches.

	Footnotes

 
¹ Supported by National Cancer Institute Grant R01CA67819 and Aventis Pharmaceuticals.

³ The abbreviations used are: CDK, cyclin-dependent kinase; pRb, retinoblastoma protein; ppRb, phospho-pRb; QFM, quantitative fluorescence microscopy.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/25/02; revised 2/18/03; accepted 3/ 4/03.

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