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[Cancer Research 59, 1492-1497, April 1, 1999]
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[Cancer Research 59, 1492-1497, April 1, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

Use of Alanosine as a Methylthioadenosine Phosphorylase-Selective Therapy for T-Cell Acute Lymphoblastic Leukemia in Vitro1

Ayse Batova, Mitchell B. Diccianni, Motoko Omura-Minamisawa, John Yu, Carlos J. Carrera, Louis J. Bridgeman, Faith H. Kung, Jeanette Pullen, Michael D. Amylon and Alice L. Yu2

Departments of Pediatric Hematology/Oncology [A. B., M. B. D., L. J. B., A. L. Y., M. O-M., F. H. K.] and Medicine 92093 [C. J. C.], University of California 92103, The Scripps Research Institute [J. Y.], San Diego, California 92037; Stanford University, Palo Alto, California 94305 [M. D. A.]; and The University of Mississippi, Jackson, Mississippi 39216 [J. P.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methylthioadenosine phosphorylase (MTAP) is an important enzyme for the salvage of adenine and methionine and is deficient in a variety of cancers including T-cell acute lymphocytic leukemia (T-ALL). Previously, we reported that the MTAP gene was deleted in over 30% of T-ALL patients at both diagnosis and relapse. We now report that MTAP- primary T-ALL cells are more sensitive to the toxicity of L-alanosine, an inhibitor of de novo AMP synthesis, than are MTAP+ primary T-ALL cells. As measured by [3H]thymidine incorporation, DNA synthesis in all seven MTAP- primary T-ALL cells was inhibited by L-alanosine with a mean IC50 of 4.8 ± 5.3 µM (range, 0.3–11.3 µM). On the other hand, the IC50 for 60% (12 of 20) of MTAP+ primary T-ALL was 19 ± 18 µM (range, 1.7–67 µM;P = 0.02), whereas the remaining 40% (8 of 20) had an IC50 of >80 µM. Furthermore, normal lymphocytes and MTAP+ primary T-ALL cells were rescued from L-alanosine toxicity by the MTAP substrate 5'-deoxyadenosine, but MTAP- T-ALL cells were not. These results indicate that normal cells, which are intrinsically MTAP+, would be protected from L-alanosine toxicity, whereas MTAP- tumor cells would be killed. Thus, our results support the use of L-alanosine alone or in combination with a salvage agent as a MTAP-selective therapy and therefore lay the foundation for the initiation of clinical trials for the treatment of T-ALL and other MTAP-deficient malignancies with L-alanosine.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ALL3 is the most common type of cancer in children. T-ALL comprises 15–20% of ALL and requires more intensive therapy than the B-cell-type ALL. An understanding of the molecular biology of T-ALL would facilitate the development of selective therapies by exploitation of the biological properties specific to T-ALL cells and thereby improve the outlook for this disease.

MTAP is an important salvage enzyme for both adenine and methionine. Specifically, MTA generated during the synthesis of polyamines is rapidly cleaved by the ubiquitous enzyme MTAP into adenine and 5-methylthioribose-1-phosphate (Fig. 1Citation ; Ref. 1 ). Adenine is efficiently salvaged to form AMP by adenine phosphoribosyltransferase, and 5-methylthioribose-1-phosphate is converted to methionine by a complex set of oxidations via the intermediate 2-keto-4-methylthiobutyrate (2) . MTAP is abundant in all normal cells including erythrocytes (3 , 4) and bone marrow stem cells (5) but is deficient in several tumor cell lines (4 , 6) and primary tumors including glioma (7) , non-small cell lung cancer (8 , 9) , acute non-lymphoid leukemia (10 , 11) , and melanoma (10) . The MTAP gene is located on chromosome 9p21, 100 kb telomeric of the genes encoding the cyclin-dependent kinase inhibitors p15 and p16, which are often deleted in tumor cells (12, 13, 14, 15, 16) . Previously, we reported for the first time that the MTAP gene is deleted in over 30% of T-ALL patients at both diagnosis and relapse and is always associated with the deletion of p16 (17) . The deficiency of MTAP in tumor cells offers a unique opportunity to develop a selective therapy that would spare normal cells. In MTAP- cells, the salvage of adenine from MTA would be blocked, resulting in an increased dependency on the de novo synthesis of adenine nucleotides. Thus, a MTAP-selective therapy can be developed in which MTAP-deficient tumor cells would be killed with drugs that block de novo AMP synthesis, under conditions in which MTAP+ normal cells would be rescued by MTA or by other MTAP substrates that would provide a source of adenine.



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  		Fig. 1. Role of MTAP in the salvage of adenine and methionine from MTA and the site of action of L-alanosine. HPRT, hypoxanthine phosphoribosyl transferase; ASS, adenylosuccinate synthase; ASL, adenylosuccinate lyase; AK, adenosine kinase; APRT, adenosine phosphoribosyl transferase; SpdS, spermidine synthetase; SpmS, spermine synthetase; SAMDC, S-adenosylmethionine decarboxylase; alanosylAICOR,L-alanosyl-5-amino-4-imidazole carboxylic acid ribonucleotide.

 
L-Alanosine is a potent inhibitor of de novo AMP synthesis (18, 19, 20, 21) . The metabolite of L-alanosine, L-alanosinyl-5-amino-4-imidazole carboxylic acid ribonucleotide, is a strong inhibitor of adenylosuccinate synthase that converts IMP to AMP. Because of its specificity for AMP, L-alanosine is an ideal candidate for MTAP-targeted therapy. In the present study, we tested the efficacy of L-alanosine in killing MTAP- primary T-ALL cells in the presence of a salvage agent to spare normal cells. Our results indicate that MTAP- T-ALL cells are more sensitive to the toxicity of L-alanosine than are MTAP+ cells. In addition, normal lymphocytes and MTAP+ primary T-ALL cells are rescued from L-alanosine toxicity, whereas MTAP- primary T-ALL cells are not.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
L-Alanosine was a generous gift from the Drug Synthesis and Chemistry Branch/Developmental Therapeutic Program/Division of Cancer Treatment (National Cancer Institute, Bethesda, MD). The 5'-deoxyadenosine was purchased from Sigma Chemical Co. (St. Louis, MO).

Patient Population and Isolation of Primary T-ALL Cells.
Heparinized bone marrow or peripheral blood samples were obtained at diagnosis from T-ALL patients who enrolled in Pediatric Oncology Group Protocols 9000 and 9400 (ALL Biology Study). The patient population was 25% female and 75% male. To safeguard patient confidentiality, each patient was assigned a code number. For normal controls, peripheral blood samples were obtained from healthy individuals.

Mononuclear cells from bone marrow or peripheral blood were isolated by isopyknic sedimentation through Ficoll-Hypaque (specific gravity, 1.077 g/ml; Pharmacia, Piscataway, NJ) at 400 x g for 30 min, followed by two washes in RPMI 1640. The content of lymphoblasts, as determined by Wright stain, was generally >= 80%. Both normal lymphocytes and primary T-ALL cells were cultured in RPMI 1640 supplemented with 10% horse serum, 2 mM glutamine, and 1% penicillin/streptomycin (complete media).

Southern Blot Analysis of MTAP in Primary T-ALL.
Genomic DNA was isolated from mononuclear cells using the Genomic DNA Isolation Kit from Life Technologies, Inc. (Gaithersburg, MD). Southern blot analysis of the MTAP gene was performed as described previously (17) .

PCR Amplification of Exon 8 of MTAP.
Semiquantitative PCR amplification of exon 8 of MTAP was performed as described previously (17) . The GAPDH gene was amplified by PCR as a control for DNA concentration, using 50 ng of genomic DNA in a total volume of 50 µl containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mm MgCl2, 100 µM deoxynucleotide triphosphate mix, 10 pmol each of sense and antisense primer, and 1.25 units of Taq DNA polymerase (Life Technologies, Inc.). After an initial denaturation at 95°C for 3 min, amplification proceeded for 1 min each at 95°C, 55°C, and 72°C for 24 cycles. A final extension was performed at 72°C for 2 min. Oligonucleotide primers for GAPDH were as follows: sense, 5'-TGGTATCGTGGAAGGACTCATGAC-3'; and antisense, 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3'.

Determination of the Sensitivity of MTAP+ and MTAP- Primary T-ALL Cells to L-Alanosine Cytotoxicity.
Primary T-ALL cells were plated at 0.5 x 106 cells/ml into 96-well plates in complete RPMI containing increasing concentrations of L-alanosine. The cells were cultured for 3 days and then pulsed with [3H]thymidine for 6 h before determining incorporation in a scintillation counter.

Determination of the Ability of a Salvage Agent to Rescue MTAP+ Normal Lymphocytes and T-ALL Cells from L-Alanosine Toxicity.
Normal lymphocytes and primary T-ALL cells were plated at 0.5 x 106 cells/ml into 96-well plates in complete RPMI containing 20 or 40 µML-alanosine and increasing concentrations of 5'-deoxyadenosine. Normal lymphocytes were stimulated to proliferate with 1 µg/ml PHA. Cells were cultured for 3 days before determining [3H]thymidine incorporation.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Determination of the MTAP Gene Status in Primary T-ALL.
To evaluate the effectiveness of therapeutic agents selective for MTAP- cells, we first determined the MTAP gene status of primary T-ALL cells and then correlated gene status with in vitroL-alanosine toxicity. In our previous study (17) , we established that there is complete agreement between MTAP gene status results obtained by Southern blot analysis and those obtained by PCR amplification. Thus, in the present study, the MTAP gene was examined in 27 T-ALL patient samples by Southern blot analysis and/or PCR amplification. The results of a representative Southern blot analysis are shown in Fig. 2Citation . Primary T-ALL samples in Lanes 1, 3, and 5–8 reveal an intact MTAP gene because all exons are present except for exon 1, which is difficult to detect by Southern analysis (12 , 13) . However, the T-ALL sample in Lane 4 (patient 127249) reveals a deletion of the entire MTAP gene, and the T-ALL sample in Lane 2 (patient 127155) reveals a partial deletion including exons 5–8. We (17) and others (13) have reported previously that a breakpoint often occurs between exons 4 and 5 of the MTAP gene, resulting in the deletion of exons 5–8. Because exon 8 is always deleted when there is a deletion in the MTAP gene, semiquantitative PCR amplification of this exon was performed to confirm and extend the results obtained by Southern blot analysis. The results of a representative PCR amplification of exon 8 of MTAP are presented in Fig. 3Citation . Dilutions of normal lymphocyte DNA with DNA from a MTAP- cell line, K562, were used in each PCR experiment as internal standards to enable semiquantitation of MTAP in primary T-ALL samples. Primary T-ALL in Lanes 6, 9, and 10 (patients 127155, 127249, and 130138, respectively) have a deletion of exon 8, based on comparison with the standard containing 10% normal lymphocyte DNA (Lane 3). T-ALL samples in Lanes 5, 7, and 8 (patients 127093, 127342, and 839, respectively) have an intact MTAP gene. These results confirm and extend the results from Southern blot analysis (Fig. 2)Citation . Overall, 7 of 27 (26%) T-ALL patients examined had a deletion of the MTAP gene.



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  		Fig. 2. Southern blot analysis of MTAP in primary T-ALL. EcoRI-digested genomic DNA (10 µg) was hybridized with a MTAP cDNA probe. The MTAP pseudogene (P) was used as a control for DNA loading. Lanes 1, 3, and 5–8, MTAP+ primary T-ALL DNA (patients 127093, 127342, 126972, 821, 795, and 126258, respectively); Lanes 2 and 4, MTAP- primary T-ALL DNA (patients 127155 and 127249, respectively). MTAP exons 1–8 are labeled accordingly.

 


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  		Fig. 3. Semiquantitative PCR amplification of exon 8 of MTAP in primary T-ALL. PCR amplification was performed with 50 ng of genomic DNA as described in "Materials and Methods." Normal lymphocyte DNA was diluted with MTAP- K562 cell DNA to generate a standard curve for the quantitation of MTAP in primary T-ALL samples. The GAPDH gene was used as a control for DNA loading. PCR products were resolved on a 2% agarose/TAE gel. Lanes 1–4, normal lymphocyte DNA at 100%, 25%, 10%, and 0%, respectively; Lanes 5, 7, and 8, MTAP+ primary T-ALL DNA (patients 127093, 127342, and 839, respectively); Lanes 6, 9, and 10, MTAP- primary T-ALL DNA (patients 127155, 127249, and 130138, respectively).

 
In Vitro Sensitivity of Primary T-ALL to the Cytotoxicity of L-Alanosine.
Because cancer cells lacking the MTAP gene are not able to salvage adenine from MTA, the natural MTAP substrate, they are more dependent on de novo synthesis. Thus, blocking the de novo synthesis of AMP with L-alanosine should result in a greater sensitivity to L-alanosine toxicity in MTAP- cells compared to MTAP+ cells. Having established the MTAP gene status of primary T-ALL cells, we examined their sensitivity to the cytotoxicity of L-alanosine and correlated the results with their MTAP status. As shown in Figs. 4Citation and 5Citation there is a significant difference between the sensitivities of MTAP- and MTAP+ cells to L-alanosine. For instance, in a representative experiment, treatment with 10 µML-alanosine resulted in a 73% inhibition of [3H]thymidine incorporation in MTAP- T-ALL cells, but only a 28% inhibition in MTAP+ T-ALL cells (Fig. 4)Citation . Heterozygous deletion of the MTAP gene did not necessarily confer an increased sensitivity to L-alanosine toxicity (data not shown). The chemosensitivity to L-alanosine of 27 T-ALL patient samples is summarized in Fig. 5Citation . The mean IC50 of L-alanosine was 4.8 ± 5.3 µM (range, 0.3–11.3 µM) in MTAP- T-ALL cells and 19 ± 18 µM (range, 1.7–67 µM; P = 0.02, Mann-Whitney test) in 60% (12 of 20) of MTAP+ T-ALL cells. The remaining 40% (8 of 20) of MTAP+ T-ALL samples were relatively resistant to the cytotoxicity of L-alanosine and had an IC50 greater than 80 µM. The variability in the sensitivity to L-alanosine toxicity among the MTAP+ T-ALL cells is most likely due to the wide range of MTAP enzyme activity present even among the same cell types (11 , 22) as well as the variation in adenylosuccinate synthase activity and, possibly, the ATP turnover rate.



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  		Fig. 4. Toxicity of L-alanosine to MTAP- and MTAP+ primary T-ALL cells. T-ALL cells were plated at 0.5 x 106 cells/ml in complete RPMI (10% horse serum, 2 mM glutamine, and 1% penicillin/streptomycin) containing increasing concentrations of L-alanosine and cultured for 3 days. Cells were then pulsed with [3H]thymidine for 6 h. •, MTAP- cells (patient 125317); {blacktriangleup}, MTAP+ cells (patient 128895).

 


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  		Fig. 5. Comparison of the IC50 of alanosine in MTAP-versusMTAP+ primary T-ALL cells. T-ALL cells were plated at 0.5 x 106 cells/ml in complete RPMI (10% horse serum, 2 mM glutamine, and 1% penicillin/streptomycin) containing increasing concentrations of L-alanosine and cultured for 3 days. Cells were then pulsed with [3H]thymidine for 6 h.

 
Selective Salvage of MTAP+ Primary T-ALL Cells from L-Alanosine Toxicity.
All normal cells possess the MTAP gene, which is essential for the salvage of adenine from the natural MTAP substrate, MTA. MTAP- tumor cells are not able to rely on this salvage pathway and are more dependent on the de novo synthesis of AMP. Thus, the use of a salvage agent in combination with L-alanosine would further protect normal cells while killing MTAP- tumor cells. To test this hypothesis, PHA-stimulated normal lymphocytes as well as both MTAP+ and MTAP- primary T-ALL cells were treated with 20 or 40 µML-alanosine in the presence of increasing concentrations of the salvage agent, 5'-deoxyadenosine. 5'-Deoxyadenosine was preferred over MTA as the salvage agent because it is less toxic to cells at higher concentrations (17) . Both PHA-stimulated normal lymphocytes (Fig. 6)Citation and MTAP+ primary T-ALL cells (Fig. 7)Citation were rescued completely or nearly completely from L-alanosine toxicity by concentrations of 5'-deoxyadenosine of less than 20 µM. For example, in the absence of 5'-deoxyadenosine, PHA-stimulated normal lymphocytes incorporated [3H]thymidine at levels of 40% and 20% of control (PHA only) when treated with 20 and 40 µML-alanosine, respectively. Upon the addition of 10 µM 5'-deoxyadenosine, [3H]thymidine uptake was restored to near (80%) control levels (Fig. 6)Citation . Similarly, in the absence of 5-deoxyadenosine, MTAP+ primary T-ALL cells incorporated [3H]thymidine at 20% of control (media alone), but upon the addition of 5 µM 5'-deoxyadenosine, incorporation of [3H]thymidine increased to control levels. On the other hand, MTAP- primary T-ALL cells were not rescued from L-alanosine toxicity by up to 20 µM 5'-deoxyadenosine (Fig. 7)Citation . The results of salvage experiments obtained with 22 T-ALL patient samples are summarized in Table 1Citation . All 17 MTAP+ samples (with either one or two alleles present) but none of 5 MTAP- T-ALL samples were rescued from L-alanosine toxicity by 5'-deoxyadenosine. Because T-ALL cells harboring a heterozygous MTAP deletion did not respond differently than those with both MTAP alleles intact, a distinction was not made between these samples. Our results indicate that in the presence of a rescue agent such as 5'-deoxyadenosine, L-alanosine can selectively kill MTAP- tumor cells. The slight increase in [3H]thymidine incorporation observed in the MTAP- T-ALL sample from patient 130138 and, to a lesser extent, in the sample from patient 127845 upon the addition of 5'-deoxyadenosine is most likely due to the presence of a small subpopulation of T-ALL cells that are MTAP+. PCR amplification of the MTAP gene (Fig. 3, Lane 10)Citation revealed that the sample from patient 130138 contained a significant amount of MTAP, although much less than that in the normal control.



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  		Fig. 6. Rescue of normal lymphocytes from L-alanosine toxicity. Normal lymphocytes were plated at 0.5 x 106 cells/ml and cultured in complete RPMI containing 1 µg/ml PHA, 20 (•) or 40 µM ({blacktriangleup}) L-alanosine, and increasing concentrations of 5'deoxyadenosine for 3 days. Control cultures received PHA alone. Cells were pulsed with [3H]thymidine for 6 h.

 


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  		Fig. 7. Rescue of MTAP+ but not MTAP- primary T-ALL cells from L-alanosine toxicity. T-ALL cells were plated at 0.5 x 106 cells/ml and cultured for 3 days in complete RPMI alone (control), or in complete RPMI containing 20 µML-alanosine and increasing concentrations of 5'deoxyadenosine. Cells were then pulsed with [3H]thymidine for 6 h. •, MTAP- cells (patient 125317); {blacktriangleup}, MTAP+ cells (patient 876).

 

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  		Table 1 Rescue of MTAP+ but not MTAP- primary T-ALL cells from L-alanosine toxicity

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To date, treatment of cancer has involved regimens that are toxic not only to cancer cells but to normal cells as well, resulting in many severe side effects in cancer patients. The existence of molecular differences between normal and tumor cells may allow the development of a tumor-selective chemotherapy in which normal cells would be spared, thus eliminating toxic side effects and increasing the therapeutic index. MTAP deficiency is one metabolic difference at the molecular level between normal and tumor cells that may be exploited for such a selective anticancer therapy. MTAP is abundant in normal cells but is deficient in many human cancer cell lines and primary tumors (3, 4, 5, 6, 7, 8, 9, 10, 11) . We have reported previously that in T-ALL, the MTAP gene is deleted in greater than 30% of patients at both diagnosis and relapse (17) . Previous studies have demonstrated that the growth of MTAP- cancer cell lines can be selectively inhibited by de novo purine synthesis inhibitors in the presence of salvage agents (4 , 9) . These studies made use of the de novo purine synthesis inhibitors methotrexate and 5,10-dideazatetrahydrafolic acid in combination with MTA and thymidine. These inhibitors are not specific for AMP synthesis and inhibit the synthesis of GMP. In addition, methotrexate inhibits the synthesis of thymidylate. Thus, rescue by MTA or other MTAP substrate would not be sufficient to completely protect normal cells from these drugs, as demonstrated by Hori et al. (23) . L-Alanosine, on the other hand, is an ideal candidate for MTAP-targeted therapy because it blocks AMP synthesis exclusively by inhibiting adenylosuccinate synthase. MTAP+ cells, whether harboring one or two alleles of MTAP, can replenish their depleted ATP pools by salvaging adenine from MTA or another MTAP substrate and completely escape toxicity, as demonstrated by our results in Figs. 6Citation and 7Citation .

Previously, little success was obtained in Phase I and Phase II clinical trials of L-alanosine in patients with leukemia and solid tumors including breast, colon, and head and neck cancers (24, 25, 26, 27) . However, these patients were not evaluated for MTAP status, and patients with malignancies known to have a high incidence of MTAP deficiency such as glioma, non-small cell lung carcinoma, and T-ALL were not included in these trials. In animal studies, however, L-alanosine significantly prolonged the life of mice bearing L1210 and P388 tumors but not that of mice bearing B16 melanoma (21) . It is of interest to note that L1210 and P388 are MTAP deficient, whereas B16 is not MTAP deficient. The study by Hori et al. (23) further demonstrates that MTAP deficiency contributes directly to the sensitivity of cancer cells to L-alanosine. In this study, the investigators transfected MTAP-deficient A549 cells with expression vectors encoding MTAP cDNA in either the sense or antisense orientation and examined the response of the transfectants to L-alanosine. The results indicated that the antisense transfectants (MTAP-) were more sensitive to L-alanosine than the sense (MTAP+) transfectants. Furthermore, MTA was able to completely rescue the sense transfectants, but not the antisense counterparts, from growth inhibition by L-alanosine. Thus, in genetically identical cells, MTAP status alone determines the response to L-alanosine. It is evident that the knowledge of MTAP status in previous clinical trials of L-alanosine would have yielded a much more successful outcome. Our present results on the selectivity of L-alanosine for MTAP- primary T-ALL, in addition to our previous report on MTAP deficiency in primary T-ALL (12) , document for the first time the validity of MTAP-targeted chemotherapy in primary cancer cells. These studies lay the foundation for the initiation of clinical trials of L-alanosine administered with and without a salvage agent for the treatment of T-ALL and other MTAP- cancers.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the excellent manuscript preparation of Greg Best. We are also grateful to the Pediatric Oncology Group Cell Bank at St. Jude Children’s Hospital (Memphis, TN) for providing some cryopreserved T-ALL samples at diagnosis.


   FOOTNOTES
 
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.

1 Supported by NIH Grants CA70391 (to A. L. Y.), U10CA28439 (to F. H. K.), and LSA6226 (to J. Y.), DHP-164 (to C. J. C.) and in part by Grants MO1 RR00827 from the General Clinical Research Center program and by the Cinyd Matters Fund. Back

2 To whom requests for reprints should be addressed, at Department of Pediatrics/Hematology Oncology, University of California San Diego Medical Center, 200 West Arbor Drive, San Diego, CA 92103-8447. Phone: (619) 543-6844; Fax: (619) 543-5413; E-mail: a1yu{at}ucsd.edu Back

3 The abbreviations used are: ALL, acute lymphocytic leukemia; T-ALL, T-cell ALL; MTAP, methylthioadenosine phosphorylase; MTA, 5'-deoxy-5'methylthioadenosine; PHA, phytohemagglutinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received 8/12/98. Accepted 2/ 3/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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