ARTICLE |
Correspondence to: Massimo Derenzini, Alma Mater Studiorum, Università di Bologna, Dipartimento di Patologia Sperimentale, Via San Giacomo 14, 40126 Bologna, Italy. E-mail: derenzin@med.unibo.it
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Summary |
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In this study we investigated the relationship between thymidylate synthase (TS) protein expression, evaluated by Western blotting analysis and by immunohistochemistry (IHC), and growth rate in human colon xenograft tumors in nude mice. Human colon cancer cell lines were used to induce xenograft tumors and the tumor mass growth rate was calculated by measuring tumor size variations over time. TS 106 monoclonal antibody was used for both Western blotting and IHC TS detection. Tumor cell growth fraction was measured by Ki67/MIB1 immunolabeling and tumor cell growth rate by evaluating the mean nucleolar size in silver-stained sections. TS Western blotting values were related to tumor mass growth rate (p<0.001) and cell growth rate (p=0.002) but not to cell growth fraction (p=0.676). The degree of the IHC staining showed only a trend to be associated with TS protein expression measured on Western blotting, and was not related either to tumor mass growth or cell proliferation rate. Tumor xenografts were also characterized for TS promoter tandem repeat and p53 status. No relationship was observed between these variables and TS expression evaluated by both Western blotting and IHC analysis. Our results demonstrate that TS expression evaluated by Western blotting analysis is directly related to the tumor mass growth rate and question the use of the IHC approach to obtain precise quantitative information on TS expression in tumor samples.
(J Histochem Cytochem 50:16331640, 2002)
Key Words: colorectal cancer, thymidylate synthase, tumor growth rate, cell kinetics, silver-stained nucleolus, Western blotting, immunohistochemistry, nude mice
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Introduction |
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Thymidylate synthase (TS) is necessary for the de novo synthesis of DNA in proliferating cells, catalyzing the methylation of deoxyuridine monophosphate to thymidine monophosphate (
Precise quantification of TS can be carried out either by evaluating the enzyme catalytic activity, by the [3H]-FdUMP enzyme binding analysis, by RT-PCR, or by Western blotting. However, these methods are difficult to use in routine tumor pathology because tumor samples frequently contain normal cells and TS values are dependent on the purity of the samples. Moreover, these methods need fresh frozen tissue and can not be applied to archival material. These drawbacks can be surmounted by immunocytochemistry (IHC), which permits discrimination between tumor and normal cells and the use of routinely fixed and embedded tissue samples. On the other hand, quantitative IHC is far from being a reliable standardized procedure. TS quantification by IHC is performed by evaluating the intensity of the staining reaction, which is subject to variables linked to the staining reaction per se, to subjective operator evaluation, and to subjective comparative evaluation. Nevertheless, the IHC approach is still the most widely used method for TS quantification in routine tumor pathology.
In a previous study (
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Materials and Methods |
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Cancer Cell Lines
Six established human colon cancer cell lines were used for the experiments: SW 480, SW 948 FL (a clone from SW 948;
TS Tandem Repeat Polymorphism Determination
DNA was extracted from the six cancer cell lines used for the study by a salting out-based method (
Xenografting Procedure
The six cell lines were xenografted in female athymic Swiss mice (nu/nu genotype; Charles River, Calco, Italy). Monolayer cultured cells were detached by trypsinization, washed in PBS, and counted. Approximately 10 x 106 cells were resuspended in 0.1 ml PBS and inoculated intradermally in the flanks of mice. Three animals per cell line were inoculated. The mice were maintained under specific pathogen-free conditions at constant temperature (2428C) and humidity (3050%). Sterilized food and tapwater were given ad libitum. The animals were examined daily for tumor growth and sacrificed by cervical dislocation after general anesthesia. The protocols used were approved by the Local Animal Care Committee. Immediately after sacrifice the formed tumors were removed and cut in two. One half was frozen in liquid nitrogen and stored at -80C, and one half was fixed in 4% buffered formalin and routinely paraffin embedded. From each paraffin block five consecutive sections were cut, which were stained with hematoxylin and eosin, silver nitrate, and MAbs anti-TS, anti-Ki67, and anti-p53.
Evaluation of Tumor Mass Doubling Time
The tumor mass doubling times (DTs) were derived by calculating the tumor volume variations over a defined period of time and applying the formula proposed by
DT=tx
where t is the time interval between measurements and V0 and V1 are the tumor mass volumes calculated 10 days after cancer cell inoculation and before sacrifice, respectively. The estimated volumes of the developing tumors were obtained by measuring the two main diameters with a caliber and applying the following formula:
tumor volume=xlengthxwidth2
Preparation of Protein Extracts for SDS-PAGE
Tumor specimens were treated by lysis buffer (KH2PO4 0.1 M, pH 7.4, 1% Igepal CA 630; Sigma Chemical, St Louis, MO) at 4C and immediately homogenized. Lysates were then incubated for 10 min on ice and then centrifuged for 45 min at 20,000 x g. The supernatant was then directly added to Laemmli buffer and boiled for 5 min. All steps were performed at 4C and all solutions contained a cocktail of complete protease inhibitors (Roche Diagnostics Italia; Milan, Italy).
Protein concentration of each lysate was assessed in triplicate by the Lowry method on aliquots collected before the adding of Laemmli buffer. Twenty micrograms of proteins from each sample were electrophoresed in 12.5% SDS-PAGE. Size standards from 200 to 29 kD, purchased from Sigma, were included in each gel. Polypeptides were electrotransferred to reinforced cellulose nitrate membranes (Hybond C Extra; Amersham, Poole, UK).
Immunoblotting
After electroblotting, filters were stained with Ponceau solution (Sigma) and then saturated with 5% non-fat dry milk, 1.5% BSA in TBS (20 mM Tris-HCl, 137 mM NaCl, pH 7.6)0.1% Tween-20 (TBS = T; Sigma) for 1 hr at room temperature (RT). Nitrocellulose membranes were then rinsed in TBS-T and incubated for 1 hr at RT with MAb TS 106 (Chemicon International; Temecula, CA) diluted 1:400 and anti-ß-actin (Santa Cruz Biotechnology; Santa Cruz, CA) diluted 1:100 in 5% non-fat dry milk, 1.5% BSA TBS-T. Membranes were washed once for 15 min and twice for 5 min in TBS-T to remove unbound antibody, and were incubated for 1 hr in the presence of horseradish peroxidase (HRP)-labeled anti-mouse antibody (Santa Cruz Biotechnology). After several washings, the HRP activity was detected using an Enhanced Chemoluminescence kit (Amersham) and was revealed on Hyperfilm ECL (Amersham). Autoradiographs were acquired with a scanner (DUOSCAN; Agfa, Morstel, Belgium) and signals quantified using a specific densitometric software (Gel-Pro analyzer, version 3.0; Media Cybernetics, Silver Spring, MD).
Immunohistochemistry
Four-µm-thick consecutive sections were cut from formalin-fixed, paraffin-embedded tissue blocks, collected on 3-ethoxy-aminoethyl-silane-treated slides, and allowed to dry overnight at 37C. Sections were waxed in xylene and rehydrated through graded concentrations of ethanol to distilled water. Sections were then immersed in 10 mM citrate buffer (pH 6.0) and processed in a thermostatic water bath for 40 min at 98C for antigen retrieval. Anti-TS (clone TS 106; Chemicon International, dilution 1:100) and anti-Ki67 (clone MIB1; BioGenex Laboratories, San Ramon, CA, dilution 1:100), and anti-p53 (clone BP53-12.1; BioGenex Laboratories, dilution 1:1800) MAbs were applied on three slides for each case, and incubations were performed overnight at RT in a humidified atmosphere. IHC was performed according to a slightly modified SABC (stretavidinbiotinperoxidase complex) method (
Nucleolar Silver Staining and Morphometric Analysis
Nucleolar silver staining was performed according to the guidelines of the International Committee on AgNOR Quantitation (
Statistical Analysis
Correlations between variables were tested using the Spearman correlation analysis. Differences between groups were evaluated by ANOVA. A p value below 0.05 was considered significant.
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Results |
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Colon Cancer Cell Lines
The six colon cancer cell lines used were characterized by different cell doubling times, ranging from 30 hr (SW 480 cell line) to 66 hr (SW 48 cell line). A previous study (
TS Tandem Repeat Polymorphism
PCR analysis was performed to define TS promoter tandem repeat status. There is evidence that the homozygous status for triple repeat (3/3) in the promoter of the gene is associate with a higher TS protein expression than the heterozygous (2/3) and the homozygous (2/2) status for tandem repeats (
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Xenografts
Each of the six cell lines was inoculated intradermally in the flanks of three different animals, for a total number of 18 mice. One mouse (xenografted with LoVo cells) died before the development of a tumor mass. Therefore, only 17 xenografts were available for the study. In two cases (xenografted with SW 480 cells) the tumor tissue was sufficient only for protein extraction.
TS Protein Expression by Western Blotting Analysis
TS protein expression was determined by immunoblotting using MAb TS 106. This antibody is the most extensively used for the quantification of TS by Western blotting analysis, resulting in values strongly related to TS activity or TS mRNA expression (
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TS Protein Expression by IHC
TS protein expression was also determined by IHC on formalin-fixed, paraffin-embedded tissue sections, using the same MAb employed for immunoblotting (TS 106). As reported by
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Immunohistochemical TS values appeared not to be significantly related to TS protein expression evaluated by Western blotting analysis. The mean Western blotting TS value progressively decreased from the 3+ (666.26 ± 305.89; range 200.61008.4) to the 2+ (428.5 ± 313.69; range 222.691052.6) and 1+ (363.84 ± 137.30; range 202.17523.28) groups. However, the differences between groups did not reach the statistical significant level of 5% (3+ vs 2+, F=1.60 and p=0.24; 2+ vs 1+, F=0.15 and p=0.71; 3+ vs 1+, F=3.30 and p=0.11).
p53 Immunohistochemistry
p53 LI ranged from 4.3% to 100%. The p53 variable was dichotomized according to a cut-off value of 10%, as reported by most of the studies in the literature (10%). Xenograft tumors with p53 accumulation were characterized by a greater TS protein expression evaluated by Western blotting analysis than xenografts with no p53 accumulation (574.84 ± 308.77 vs 321.84 ± 152.54). However, the difference between the two groups was not statistically significant (p=0.111).
Evaluation of Cell Kinetic Parameters
The tumor mass DTs of the 17 xenografts had a mean value of 7.69 ± 6.21 (SD) days, ranging from 2.8 days in a xenograft derived from the SW 480 cell line to 23 days in a xenograft derived from the LoVo cell line. We have quantitatively evaluated the two main variables that influence the tumor mass growth rate: the percentage of cycling cells and the speed of cell proliferation. The assessment of cycling cells was obtained by MIB1 immunostaining. The MIB1 LIs of the 17 xenograft tumors had a mean value of 77 ± 15.7% (SD), ranging from 45.7% to 92.8% (Table 1). No significant correlation was found between the MIB1 LIs and the corresponding mass DTs (r=0.257; p= 0.335) indicating that in this experimental model the tumor growth rate was independent on the number of cycling cells. Fig 4 shows the MIB1-immunostained sections of two xenografts characterized by the same growth rate (7.25 days for the SW 48/b xenograft reported in Fig 4a and 7.23 days for the SW 948 FL/b xenograft reported in Fig 4b) but by a different percentage a cycling cells (90.2% in the case reported in Fig 4a and 45.7% in the case reported in Fig 4b).
The rapidity of cell proliferation was evaluated by measuring the size of the silver-stained nucleoli, which represents a well-established marker of cell growth rate (
Correlation Among TS Expression and Kinetic Parameters
TS Western blotting values were found to significantly correlate with both the tumor mass DT and to the mean nucleolar size (r=-0.784; p<0.001 and r= 0.743; p=0.002, respectively), whereas no significant correlation was demonstrated between TS values and MIB1-LIs (r=0.118; p=0.676).
TS immunostaining was not related to the tumor mass DT. The mean DT values were 6.63 ± 4.30 days in the 3+ group, 10.88 ± 9.16 days in the 2+ group, and 6.14 ± 2.38 days in the 1+ group, with the following ANOVA values: 3+ vs 2+, F=0.90 and p=0.37; 2+ vs 1+, F=0.99 and p=0.45; 3+ vs 1+, F=0.04 and p=0.84.
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Discussion |
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The present results demonstrate that TS protein expression, as evaluated by Western blotting analysis, was related to the tumor mass growth rate in human colon carcinoma xenografts in nude mice. This relationship was not found when TS protein expression was evaluated by IHC. In both technical approaches, the specific TS 106 MAb was used.
Regarding the Western blotting analysis, the TS integrated OD values of the autoradiographic signals corrected by ß-actin values directly correlated with the tumor mass growth rate. Because tumor mass growth rate depends mainly on the number of cycling cells (cell growth fraction) and the rapidity of cell proliferation (cell growth rate), we also investigated the relationship between TS protein expression and these two parameters of tumor mass growth rate. First, the possibility that the TS value variations observed in our experimental model might have been related to differences in the number of cycling cells was considered. Indeed, remarkable quantitative changes of TS protein expression have been reported to occur when non-proliferating cells are stimulated to proliferate or when proliferating cells are induced to pass from the exponential to the confluent growth phase (
The possibility has been considered that the different TS protein quantitative expression observed in the xenograft tumors might be related either to TS gene promoter status (
The demonstrated strict relation between TS protein expression, as evaluated by Western blotting analysis, and cell proliferation rate enabled us to use the human colon cancer xenografts, whose growth rate was precisely measured, for assessing the reliability of IHC to obtain information on TS protein expression in tissue sections. TS IHC analysis is the method most commonly used for the evaluation of TS expression in routine tumor pathology to obtain information on the prognosis of tumor disease and, in the case of gastrointestinal cancers, indication of clinical response to 5-FU-based chemotherapy. However, contradictory results on the relationship between TS expression, evaluated by IHC, and tumor prognosis and response to chemotherapy have been produced on the same type of cancer lesions (
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Acknowledgments |
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Supported by grants from the Ministero della Ricerca Scientifica e Tecnologica (MURST) 40% and 60%, Pallotti's Legacy for Cancer Research, and University of Bologna (funds for selected research topics).
Received for publication February 6, 2002; accepted June 19, 2002.
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