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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 1 302-308
Copyright © 2000 by The Endocrine Society

Original Studies

Effects of Exogenous p53 Transduction in Thyroid Tumor Cells with Different p53 Status1

F. Moretti, S. Nanni, A. Farsetti, M. Narducci, M. Crescenzi, S. Giuliacci, A. Sacchi and A. Pontecorvi

Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute (F.M., S.N., A.F., M.N., M.C., S.G., A.S., A.P.); Institute of Experimental Medicine, Consiglio Nazionale delle Ricerche (F.M., A.F.); Department of Endocrinology, University La Sapienza (M.N.); Laboratory of Compared Toxicology and Ecotoxicology, ISS (M.C.); and Institute of Medical Pathology, Catholic University (A.P.), 00158 Rome, Italy

Address all correspondence and requests for reprints to: Alfredo Pontecorvi, M.D., Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute and Institute of Medical Pathology, Catholic University, Via delle Messi d’Oro 156, 00158 Rome, Italy.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recovery of p53 function in undifferentiated thyroid carcinoma cells carrying an altered p53 gene is able to modify cell tumorigenic properties. It is not known whether such an effect may also be achieved in thyroid cancer cells expressing wild-type p53, as in the majority of differentiated thyroid carcinomas. Effects of p53 transduction in a thyroid carcinoma cell line (FRO) exhibiting a wild-type endogenous p53 gene, in comparison to a cell line (WRO) exhibiting mutant p53, were investigated by using an inducible chimeric construct containing human p53 complementary DNA fused to the ligand binding domain of the estrogen receptor (p53ER). FRO cells were unaffected by exogenous p53 expression in terms of both proliferation and viability. On the contrary, p53 reexpression in WRO cells containing hemizygous mutated p53 allele caused a strong growth inhibition due to cell accumulation in the G1 phase of the cell cycle. In addition, exogenous p53 did not influence FRO cell behavior in response to TSH treatment or modify cell resistance to the chemotherapeutic agent, doxorubicin. Our results indicate that exogenous expression of wild-type p53 affects thyroid tumorigenic properties only in cells carrying an altered p53, whereas it is ineffective in cells expressing wild-type p53 activity. Therefore, the endogenous p53 status seems to be a major determinant for the effectiveness of a p53-based gene therapy for thyroid cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECOVERY OF wild-type p53 (wt-p53) tumor suppressor function in cancer cells characterized by mutations or deletions of the endogenous p53 gene is able to modify cell growth, cytotoxicity, and/or tumorigenicity (1, 2, 3). For this reason, exogenous wt-p53 transduction is being evaluated as a promising therapeutic tool for treatment of several types of human neoplasia (4, 5). Few reports have investigated the effect of a similar approach in cells carrying the wt-p53 gene. In nontransformed cell lines containing the wild-type tumor suppressor gene, p53 overexpression does not cause evident phenotype modifications (1, 6, 7, 8). In transformed cell lines containing wt-p53, exogenous p53 transduction has been reported to influence cell growth and/or differentiation (9, 10). However, in the latter system, it is not clear whether impairment of endogenous p53 function other than gene alteration is present. There is, in fact, increasing evidence that a large number of molecular events may regulate p53 activity at the posttranslation level as well as through interaction with other cellular proteins (i.e. MDM2 and MDMX) (11, 12, 13, 14, 15, 16, 17). Indeed, increased intracellular levels of MDM2 have been reported in some human tumors containing wt-p53, suggesting a pathogenic role in inactivating p53 function (18, 19).

In human thyroid tumors, p53 mutations have been described almost exclusively associated with the anaplastic histotype, but are only rarely detected in differentiated follicular and papillary carcinoma (20, 21, 22, 23, 24). At present, it is not known whether thyroid tumors carrying the wt-p53 gene exhibit alterations of p53 activity by mechanisms other than gene mutation/deletion. Several reports have shown that reexpression of wt-p53 function in anaplastic thyroid tumor cell lines exerts tumor suppressor activity and may partially revert the undifferentiated phenotype (25, 26, 27, 28). No studies have investigated the effects after exogenous p53 transduction in thyroid tumor cell lines expressing endogenous wt-p53.

In this report we have investigated whether the endogenous p53 status may influence the effectiveness of exogenous wild-type p53 transduction in reducing tumorigenic properties of thyroid tumor cells. Our results indicate that after exogenous p53 expression, no modification of cell proliferation or response to chemotherapeutic drugs could be observed in thyroid tumor cells containing wt-p53, whereas a strong tumor suppressor effect was evident in cells carrying altered p53 genes. Therefore, the endogenous p53 status seems to represent the limiting factor for the potential application of a p53-based gene therapy in the treatment of thyroid tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures, plasmids, and transfections

Human thyroid carcinoma cell lines FRO and WRO were cultured at 37 C in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Life Technologies, Inc., Gaithersburg, MD), 100 U/mL penicillin (Life Technologies, Inc.), 100 mg/mL streptomycin (Life Technologies, Inc.), and 2 mmol/L L-glutamine (Life Technologies, Inc.). WRO cells, derived from a follicular thyroid carcinoma, are characterized by the deletion of one p53 allele and by the presence, on the other allele, of a C:G to T:A transition at codon 223, resulting in the substitution of a proline with a leucine residue (22). FRO cells, derived from a poorly differentiated follicular thyroid carcinoma, were characterized by the presence of wild-type alleles for exons 5–8 (22).

The retroviral vector pBabe Purop53ER G525R (29) encodes for a wild-type p53 complementary DNA (cDNA) fused to the mutated ligand-binding domain of the estrogen receptor {alpha} under the control of the retroviral long terminal repeat promoter, and for the selectable marker puro under the control of simian virus 40. The product of the fusion gene, the chimeric protein p53ER, is expressed constitutively, but is activated to provide wild-type activity upon addition of the estrogen antagonist 4-hydroxytamoxifen (OHT) (30). The retroviral vector pBabe PuroER, generated by excision of p53 cDNA after digestion of the pBabe Purop53ER G525R at BamHI sites, was used as control. Approximately 2 x 106 exponentially growing FRO and WRO cells were stably transfected with 10 µg of each vector by electroporation (0.25 V, 950 µF for FRO cells; 0.22 V, 925 µF for WRO cells) using a Gene Pulser apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). After 12 h of culture, medium was replaced with fresh medium containing 2 µg/mL puromycin (Life Technologies, Inc.) for FRO and 1.5 µg/mL for WRO cells.

Transient transfection of thyroid carcinoma cell lines was performed by the calcium phosphate precipitation technique using the following constructs: plasmid PG13CAT, carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the polyoma virus minimal promoter and 13 copies of the p53 consensus-binding sequence; and plasmid MG15CAT carrying the CAT reporter gene driven by the polyoma virus minimal promoter and 15 copies of a mutated p53 binding site (31). WRO and FRO cells (2 x 105) were transfected in 60-mm plates with aliquots of precipitates containing 10 µg PG13CAT or MG15CAT or simian virus 40-CAT reporter plasmids and 1 µg CMVß-gal plasmid as an internal control for transfection efficiency. After 16 h from transfection, medium was replaced with fresh medium containing OHT (10-7 mol/L) or the equivalent amount of the ethanol solvent either alone or in the presence of TSH, at a final concentration of 10 mU/mL. Doxorubicin was added after 48 h at a final concentration of 0.5 µg/mL. Cells were harvested 72 h after transfection, and CAT and ß-galactosidase activities were assayed on whole cell extracts, as previously described (26). Each dish was also assayed for cell protein content (Protein Assay, Bio-Rad Laboratories, Inc.).

Proliferation rate and cell cycle analysis

The cell proliferation rate was assessed by determining cell number in a Thoma’s hemocytometer, using trypan blue exclusion as cell viability test. FRO and WRO cells were plated at a density of 7 x 104 cells/60-mm dish. Cell cycle profile was evaluated by fixing 5 x 105 cells in cold acetone-methanol (1:3) for 30 min at 4 C and staining DNA with 50 µg/mL propidium iodide in phosphate-buffered saline supplemented with 1 mg/mL ribonuclease A, for 30 min at room temperature. Cellular DNA content was measured by an EPICS XL analyzer (Coulter Corp., Miami, FL). The percentage of cells in the different cell cycle compartments was estimated by applying a mathematical histogram, based on the maximum likelihood approach.

Western blot analysis

Exponentially growing cells were cultured in 100 mm dishes. 1 x 106 cells were washed twice with phosphate-buffered saline (Life Technologies, Inc.) and resuspended in 100 µL lysis buffer [62.5 mmol/L Tris (pH 6.8), 2% SDS, 10% glycerol, 50 mmol/L dithiothreitol, and 0.1% bromophenol blue]. Cells were sonicated for 10 s and heated to 95 C for 5 min. Fifteen microliters of lysate were loaded onto a 12% SDS-polyacrylamide gel, electrophoresed, and then electroblotted onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.). Filters were blocked for nonspecific reactivity by incubation in 5% nonfat dry milk dissolved in TBST [10 mmol/L Tris-HCl (pH 7.8), 150 mmol/L NaCl, and 0.05% Tween-20] for 1 h at room temperature, and then probed with anti-p53 FL-393 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or with anti-cdc2 C-19 (Santa Cruz Biotechnology, Inc.), diluted in TBST at a concentration of 0.1 µg/mL, for 2 h at room temperature under gentle rocking. Immunoreactivity was determined using the enhanced chemiluminescence reaction (Amersham Pharmacia Biotech, Arlington Heights, IL).

Proteasome inhibition assay

Exponentially growing cells were seeded at 60% confluence in 100-mm dishes. Twenty-four hours later, the proteasome inhibitor MG132 (Z-Leu-Leu-Leu-H; Sigma) in dimethylsulfoxide (DMSO) was added directly to the culture medium at a final concentration of 0.01 mmol/L. Control cultures were exposed to an equivalent volume of DMSO. Protein extracts were prepared as described in Western blot analysis.

In vitro chemosensitivity

Cells were seeded at a density of 1.5 x 105/60-mm dish. The following day, medium was replaced with or without OHT (10-7 mol/L), and 8 h later, cells were treated with various concentrations of doxorubicin (10 ng/mL to 10 µg/mL). Cell viability was determined after 2 days. Data were calculated as the percentage of viable cells relative to solvent-treated control cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of endogenous p53 in thyroid tumor cell lines

To verify the endogenous wt-p53 status, the complete p53 messenger ribonucleic acid sequence of FRO cells was analyzed. No mutations were identified over the entire p53 cDNA sequence. To assess endogenous p53 protein expression (Fig. 1AGo), Western blot analysis was performed on total cell lysate. FRO cells expressed low p53 levels in accord with the presence of the wild-type gene. In comparison, higher p53 levels were present in WRO cells, due to the increased stability of the mutant p53 (32, 33).



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  		Figure 1. Expression of endogenous p53 and exogenous p53ER in thyroid carcinoma cells. A, Western blot analysis of endogenous p53 using the anti-p53 FL-393 antibody. Migration of the endogenous p53 protein is indicated by the arrow. B, Effects of the proteasome inhibitor MG132 on endogenous p53 levels in FRO cells. The relative intensities of the bands were quantitated by densitometry, normalized to the cdc-2 protein signal, and values were used to calculate ratios (subscribed number) in MG132-treated vs DMSO-treated cells. C, Expression of exogenous p53ER in FRO cells. Lower arrow, The endogenous p53; upper arrow, the 94-kDa chimeric p53ER. The positions of molecular markers (New England Biolabs, Inc., Beverley, MA) are indicated on the right. D, Endogenous p53 trans-activation activity in FRO cells, transfected with a wt-p53 responsive promoter/reporter construct (PG13CAT) or mutated (MG15CAT), respectively, and after induction by doxorubicin (0.5 µg/mL). Results represent the average ± SEM of five independent experiments, each performed in duplicate.

 
Trans-activation properties of endogenous p53 were evaluated in transient transfection assay using a CAT reporter gene driven by a promoter containing multiple p53-responsive elements (PG13CAT). A similar construct containing mutated recognition sequences unable to bind p53 (MG15CAT) was used as control The results showed that FRO-p53 is transcriptionally active on the PG13CAT vector, but not on the mutated promoter (Fig. 1DGo).

In addition to its basal transcription function, FRO-p53 was also able to respond to external stimuli, such as doxorubicin, which is a known inducer of p53 transcription activity (34) and which, at a concentration of 0.5 µg/mL, caused a 2-fold increase in p53 activity (Fig. 1DGo).

Results obtained in FRO cells are in contrast to those from other studies (35, 36, 37) that reported very low or undetectable p53 protein levels. For this reason we tested whether in our cell system, p53 was correctly processed through the proteasome pathway. In the presence of the MG132 proteasome inhibitor, p53 accumulated within the cell with respect to the cdc-2 protein, which is known not to be degraded via the proteasome pathway (Fig. 1BGo) (38). These data indicate that the known proteasome degradation pathway of p53 was still active in FRO cells.

Expression and trans-activation properties of exogenous p53 (p53ER) in thyroid tumor cells

FRO cells were stably transfected with an expression plasmid containing the human p53-coding sequence fused to a modified carboxyl-terminus, hormone-binding domain of the estrogen-receptor {alpha} (p53ER). This construct produces a p53 chimera whose activity can be stimulated by OHT (30, 39, 40).

Expression of p53ER was first evaluated by Western blot analysis, followed by indirect immunofluorescence to monitor for correct intranuclear localization of the chimeric protein. FRO-p53ER cells showed a band, which was absent in controls, of approximately 94 kDa, which is the expected size for the exogenous chimeric protein (39) (Fig. 1CGo). Indirect immunofluorescence confirmed the presence of the p53ER and its correct nuclear localization (data not shown), as also reported by others (30, 39).

To test the transcriptional activity of p53ER, transient transfection assays were performed using the p53-responsive reporter vectors (PG13CAT and MG15CAT) described above. Mock-transfected FRO cells (FRO-pBER) showed significant p53 activity due to the presence of the endogenous wild-type protein (Fig. 2AGo). No additional activity was observed in FRO-p53ER cells after OHT treatment. These results indicate that in FRO cells, p53ER, despite its intracellular expression, did not confer enhanced trans-activation over endogenous p53 activity. To test the activity of the chimeric construct, we transfected WRO cells, characterized by endogenous mutant p53, with the same plasmids (pBER and p53ER). WRO-p53ER cells showed an increase of about 2-fold over endogenous p53 activity (Fig. 2BGo), and OHT treatment further induced p53 trans-activating function (~2-fold).



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  		Figure 2. Exogenous p53ER transcriptional activity in thyroid carcinoma cells. p53 trans-activation in FRO (A) and WRO (B) cells, stably transfected either with pBER or p53ER constructs, was assessed on a wt-p53-responsive (PG13CAT) or a mutated promoter/reporter construct (MG15CAT). Results represent the average ± SEM from three to five independent experiments, each performed in duplicate.

 
These results confirm that p53ER exhibits wild type-like activity and can be induced by OHT, although the chimeric protein is already transcriptionally active in the absence of the inducer (30).

Effects of exogenous p53 on thyroid tumor cell proliferation

We have previously demonstrated that restoration of p53 function is able to strongly inhibit ARO cell proliferation (26). We therefore checked whether p53ER is also able to modify the proliferation rate of the thyroid tumor cell lines FRO and WRO depending on the status of the endogenous p53. To this purpose, cell proliferation curves and cell cycle analysis by flow cytometry were performed.

Experiments in FRO cells did not show any significant difference in proliferation rate between mock-transfected FRO-pBER and FRO-p53ER cells (Fig. 3AGo). Cell cycle analysis on day 13 of the proliferation curve confirmed these data (Fig. 3Go, B and C) showing no significant difference in the percentage of cells residing in each cell cycle phase among FRO-pBER and FRO-p53ER cells. These data are in agreement with previous experiments aimed at assessing p53 transcription function that indicated the absence of additional activity by exogenous p53ER over the endogenous protein.



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  		Figure 3. Proliferation rate and cell cycle analysis of transfected thyroid carcinoma cells. Proliferation curves of FRO (A) and WRO (D) cells transfected with pBER or p53ER constructs in either the absence or presence of OHT (10-7 mol/L) are shown. The results in A represent the average ± SEM of three independent experiments, each performed in duplicate; the results in D are representative of a typical experiment of three independent experiments. The DNA contents of FRO-pBER, FRO-p53ER, WRO-pBER, and WRO-p53ER cells, grown in the presence of OHT (10-7 mol/L), were measured by propidium iodide staining. The percentage of cells in each cell cycle phase is indicated. Results are representative of a typical experiment of three independent experiments, each performed in duplicate.

 
In WRO-p53ER cells a strong inhibition of cell growth was observed compared to that of controls (Fig. 3DGo). Dead cell counting excluded that this effect was due to an increase in cell death (data not shown). Addition of OHT did not cause further inhibition of cell proliferation. Cell cycle analysis performed on day 6 of the proliferation curve showed a strong accumulation of WROp53ER cells in the G1 phase of the cell cycle with a concomitant decrease in the S phase (Fig. 3Go, E and F).

These results indicate that exogenous p53 transduction in thyroid carcinoma cells containing an altered p53 (WRO) causes a strong inhibition of cell proliferation, with p53-mediated accumulation in the G1 phase of the cell cycle. On the contrary, proliferation of thyroid carcinoma cells carrying wt-p53 (FRO) is unaffected by exogenous p53 transduction.

Effects of exogenous p53 on thyroid tumor cell responsivity to TSH

FRO and WRO thyroid carcinoma cells exhibited growth behavior independent from TSH, the specific growth factor for thyrocytes. As it has been reported that wt-p53 is able to restore responsiveness to cytokine and hormone signals (26, 41, 42), we investigated whether p53ER transduction modifies the TSH responsiveness of the two thyroid carcinoma cell lines. In the presence of TSH (10 U/mL), an increase in the proliferation rate of WRO and FRO thyroid tumor cells was observed (Fig. 4Go, A, and B) without significant differences between p53ER-transfected cells and controls.



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  		Figure 4. Effects of TSH on the proliferation rate of thyroid carcinoma cells. FRO and WRO cells were cultured in the presence of OHT (10-7 mol/L) in the absence or presence of TSH (10 mU/mL). The results in A represent the average ± SEM of three independent experiments, each performed in duplicate; the results in B represent the average of duplicate dishes from a typical experiment of a total of three independent experiments.

 
These results indicate that FRO and WRO cells, although independent from TSH for their growth, are still stimulated to proliferate by TSH and that this hormone action is not affected by exogenous p53 expression.

Effects of exogenous p53 on thyroid tumor cell sensitivity to doxorubicin

Despite expression of exogenous chimeric p53 protein, FRO-p53ER cells did not modify their proliferation rate and/or viability. Therefore, we checked whether p53ER was able to modify FRO cell behavior under particular conditions, such as during treatment with the chemotherapeutic drug doxorubicin. After addition of doxorubicin at concentrations ranging between 10 ng/mL and 10 µg/mL, no significant modification of the IC50 between FRO-p53ER and control FRO-pBER cells was observed in either the absence or presence of OHT (Table 1Go). These results indicate that exogenous p53ER was not able to modify the chemosensitivity of the FRO cell line to doxorubicin.


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  		Table 1. In vitro chemosensitivity of FRO cells to doxorubicin

 
To better understand whether the absence of p53ER function in FRO cells could be due to inactivation of the chimeric protein, transient transfections using p53-responsive CAT reporter vectors (see above) were performed in the presence or absence of doxorubicin. In the presence of the chemotherapeutic drug, p53 transcriptional activity in FRO-p53ER cells treated with OHT was increased about 2-fold compared to that in controls (Fig. 5Go).



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  		Figure 5. Induction of p53 transcriptional activity by doxorubicin. FRO-pBER and FRO-p53ER cells were transiently transfected with mutated (MG15CAT) and wt-p53-responsive promoter/reporter constructs (PG13CAT), cultured for 72 h in the absence or presence of OHT (10-7 mol/L), and treated with Dox (0.5 µg/mL) for 24 h before analysis. Results represent the average ± SEM of three independent experiments, each performed in duplicate.

 
These data indicate that in FRO cells, p53ER, although inactive in terms of antiproliferative and transcriptional functions, is still able to respond to a trans-activation stimulus (i.e. doxorubicin) after OHT induction. Nevertheless, this effect is not sufficient to modify cell chemosensitivity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we investigated whether the endogenous p53 status of thyroid carcinoma cells influences the effectiveness of exogenously transduced wt-p53 in exerting its tumor suppressor function. An OHT-inducible p53 chimeric protein, derived from fusion of p53 cDNA with the ligand-binding domain of estrogen receptor {alpha}, was stably transfected in thyroid tumor cell lines characterized by a different p53 genetic background. In agreement with our previous results in ARO anaplastic thyroid carcinoma cells (26), transduction of wt-p53 into cells carrying an altered p53 gene was able to restore p53 tumor-suppressive function. In fact, WRO follicular thyroid carcinoma cells carrying a hemizygous mutated p53 allele after transfection with p53ER showed an almost complete arrest of proliferation with accumulation of cell population in the G1 phase of the cell cycle and concomitant reduction of the S phase. These effects were also evident in the absence of the inducer OHT, indicating that the chimeric p53 protein was active even in the uninduced state. On the contrary, FRO cells, characterized by the presence of a functional wt-p53, did not show significant modification of cell growth or increased chemosensitivity to doxorubicin after p53ER transduction in either the absence or presence of OHT.

Our data differ from other reports that demonstrated tumor suppression activity by exogenously transduced p53 in tumor cell lines containing endogenous wt-p53 (9, 10). However, these studies did not investigate endogenous wt-p53 function, limiting their characterization to the assessment of p53 messenger ribonucleic acid sequence and/or intracellular protein level. In addition to p53 gene mutations, which represent the most frequent genetic alteration detected in human cancer, increasing evidence has shown that p53 function may be modulated at multiple levels. It has been shown that phosphorylation and acetylation are key posttranslation events that intervene in regulating p53 function (12, 13, 43). p53 activity may also be modulated by interaction with other cell proteins, such as MDM2 (14, 15, 16, 43) and the recently identified MDMX (17), an MDM2 homolog that is able to inhibit wt-p53-mediated transcription. Therefore, besides the presence of the wt-p53 gene, a complex array of events may affect overall p53 function. In this respect, previous results demonstrating a tumor-suppressive function by exogenously transfected p53 in an apparent wt-p53 cell environment could be ascribed to incomplete function of the endogenous protein.

In this study we demonstrate that the thyroid tumor FRO cell line contains a wt-p53 protein that, in basal conditions, is transcriptionally active and can be further induced in response to external stimuli, such as treatment with the chemotherapeutic drug, doxorubicin. Likewise, FRO-p53 is correctly processed via the cytoplasmic proteasome system, as it accumulates into the cell after treatment with the proteasome inhibitor MG132. Transfection of the p53ER construct into FRO cells resulted in the expression of the chimeric protein, but failed to further increase p53-mediated trans-activation even in the presence of the OHT inducer. Interestingly, the apparent transcriptional inactivity of p53ER was reverted by doxorubicin treatment, which stimulated a p53-responsive CAT reporter over levels obtained by endogenous p53. Therefore, these data suggest that FRO cells maintain an intracellular machinery that controls overall p53 function.

The present study also investigated the possible interference between p53 and TSH activity and demonstrated that p53ER is not able to modify TSH activity on proliferation in both cell lines.

Gene therapy approaches based on wt-p53 transduction are currently being applied in clinical trials for the treatment of several types of human malignant neoplasia (10, 44, 45). Our results suggest that the p53 allele status represents one of the main determinants for the effectiveness of a p53-based gene therapy of thyroid tumors; this approach has less chance of being successful in the majority of differentiated thyroid tumors expressing normal p53 function.


   Acknowledgments
 
We thank Dr. Littlewood for the generous gift of p53ER plasmid.


   Footnotes
 
1 This work was supported by research grants from Associazione Italiana per la Ricerca sul Cancro, Consiglio Nazionale delle Ricerche, Ministero dell’Università e della Ricerca Scientifica e Tecnologica, A.I.R.C., C.N.R., M.U.R.S.T, and Ministero della Sanità. Back

Received June 16, 1999.

Revised September 15, 1999.

Accepted September 29, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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