Tohoku
J. Exp.
Med., 1992,
Thymidine Adenomatous
168, 291-301
Kinase
Activity
in Familial
Polyposis
SHINOBUSAKAMOTO and RYOHEI OKAMOTO Department of Function Disorder Research, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113
SAKAMOTO, S. and OKAM0T0,R. Thymidine Kinase Activity in Familial Adenomatous Polyposis. Tohoku J. Exp. Med., 1992, 168(2), 291-301 Thymidine kinase (TK) activity of polyp tissue from patients with familial adenomatous polyposis (FAP) was measured and compared with that of normal colon, sporadic polyp and colorectal carcinoma tissues. Total TK activity in colonic carcinoma was 3-fold that of normal ; this increase seems attributable mainly to increased activity of cytosolic TK isozyme activity ; the colorectal TK isozymes were separated into two types, i.e., fetal type and adult type isozymes predominantly in, respectively, cytosolic and mitochondrial fractions, by DEAEcellulose column chromatography. However, FAP polyp samples from 15 patients showed an average elevation of only 1.8-fold over normal. Examined individually, only 5 of the 15 FAP samples showed significant elevations in total TK activity. Furthermore, TK isozy u analysis revealed variable patterns of the cytosolic isozyme activity being elevated in some cases (8 of 15) and remaining low in others. Thus FAP polyps seem to be a heterogenous population with respect to DNA replicative activity, and cytosolic TK isozyme activity may constitute a biochemical marker for the subsequent development of colorectal carcinoma in FAP. familial polyposis coli (FPC) ; tymidine kinase (TK) ; isozyme
Familial adenomatous polyposis (FAP) is a genetic disorder characterized by the development of numerous adenomatous polyps in the colon and rectum. This condition is associated with a high incidence of colorectal carcinoma. A genetic locus for FAP has been identified (Bodmer, et al. 1987) on chromosome 5q; however, the structure and function of this gene remain unknown. Furthermore, other genetic alterations have also been described in the multi-step process of colorectal tumorigenesis in FAP, including loss of heterozygosity on chromosomes 17p and 18q (Okamoto et al. 1988), alterations in the expression of genes such as c-myc and cfos (Sugio et al. 1988) and the suppression of genes such as the wild-type p53 gene (Finlay et al. 1989), and mutations leading to activation of ras genes (Sasaki et al. 1990). The precise roles of these genetic alterations in the sequence of events leading to neoplasia and malignant transformation, and the corresponding alterations in the biochemistry of tumor cells, have yet to be elucidated (Fearon and Vogelstein 1990). Addressfor reprints : 1-5-45 Yushima,Bunkyo-ku, Tokyo 113,Japan. 291
292
S. Sakamoto
and R. Okamoto
We have long been using isozymes of thymidine kinase (TK ; EC 2.7.1.21), a DNA-synthesizing enzyme (Kit 1978), to examine such issues in general ; using this tool, we have recently focused on FAP in particular. Though abnormal nucleotide metabolism has previously been observed and implicated in the development of colonic cancer (Dutrillaux and Mulevis 1986; Luccioni et al. 1988), little is known about nucleotide synthesis in FAP patients. In the present study, we report the changes we have observed in the activity of thymidine kinase and its isozymes in colorectal tumors from patients with colonic cancer, with sporadic adenomatous polyps, and with FAP. MATERIALS AND METHODS Patients and materials Samples of normal colonic mucosa and colonic carcinoma were obtained from 60 patients [29 males, average age 65.2±2.0 years (range : 51-89 years), and 31 females, average age 63.1±2.3 years (range : 35-89 years)] who underwent surgical resection at the 2nd Department of Surgery, Tokyo Medical and Dental University, Japan. Upon resection, the tissues were immediately placed on ice and transferred from the operating room to the laboratory within 30 min. While on ice, the neoplastic lesions were separated from normal mucosa, and both specimens were divided into two parts, one for histology and another for enzymatic assays. The latter samples were stored at -80°C. Sporadic adenomatous polyps were resected from 15 patients [7 males, 8 females ; average age 49.6± 4.4 years (range : 14-65 years)], and the tissue specimens were immediately placed on ice and divided as described above. FAP polyp tissues were also obtained from 15 patients [9 males, 6 females ; average age 36.7±2.8 years (range : 22-52)], who underwent surgical resection for FAP. The diagnosis was established prior to resection by family history, as well as physical, radiologic, and rectosigmoidoscopic examination, and confirmed at the time of operation. These samples were also processed as described above. Preparation of enzyme extract and isolation of TK isozymes A crude enzyme extract was first prepared by pulverizing the tissue specimens with an autopulverizer under liquid nitrogen, followed by homogenization at 0°C with 10 volumes of buffer containing 5 mM Tris-HC1(pH 7.5)/0.1 mM EDTA/1 mM mercaptoethanol/0.25 M sucrose, and then collecting the supernatant after centrifugation at 105,000 g for 1 hr at 4°C. This crude extract was then used for assay of total TK activity, as described below, or fractionated by adding saturated ammonium sulfate to 50% saturation and stirring for 1 hr, followed by centrifugation at 10,000 g for 20 min. The precipitate was then resuspended in buffer containing 5 mM Tris-HC1 (pH 7.5)/20% glycerol/1 mM MgC12,and subjected to overnight dialysis against the same buffer, followed by centrifugation of the dialysate at 10,000 g for 30 min. The resultant supernatant was loaded onto a DEAE-cellulose (DE-52, Whatman, Kent, UK) column (1.5 by 5.0 cm) that had been equilibrated with the same buffer, and eludet stepwise from the column with 6 ml volumes of the same buffer containing increasing concentrations of NaCI (0 M, 0.1 M, 0.2 M, and 0.3 M). Fractions of 1.0 ml were collected and used for assay of TK isozyme activity, described below. Assay of thymidine kinase (TK) activity As previously reported, TK activity was measured by the method of Taylor et al. (1972). An assay mixture consisting of 5 mM MgC12,10mM ATP, 2pM [6 3H] thymidine, and 0.1 M Tris-HC1 buffer (pH 7.5) in a total volume of 200 p1 was incubated with either the crude enzyme preparation (for determination of total TK activity), or with fractions eluted
TK Activity
in Familial
Adenomatous
Polyposis
293
from the DEAF-cellulose column (for determination of TK isozyme activities), for 15 min at 30°C, and boiled to stop the enzyme reaction for 3 min, followed by centrifugation at 10,000g for 10 min. A 100,u1aliquot of the supernatant was spotted onto DEAE-cellulose paper, which was then washed extensively with 1 mM ammonium formate and ethanol to remove all unphosphorylated substrate, and dried ; the radioactivity remaining on the paper was then measured in a liquid scintillation counter. Characterization of biochemicalproperties of colonic TX isozymes Differential inhibitory effect of pyrimidine nucleotides on the TK isozymes was demonstrated by including deoxythymidine triphosphate (dTTP) at a final concentration of 1.0 mM, or deoxycytidine triphosphate (dCTP) at final concentrations of 0.5 mM, 1.0 mM, and 5.0 mM, in the reaction mixture. The molecular weights of the colonic TK isozymes was estimated by HPLC with use of a TSK-G3000SW column (600 by 7.5 mm internal diameter, Toyo Soda, Tokyo). The column was eluted with 0.1 M phosphate buffer containing 0.1 MNaOl (pH 6.5) at a flow rate of 0.8 ml/min. The effects of pH on the isozyme activities was studied by varying the pH of the reaction buffer with 0.1 M Tris-HCl (pH 7.0-9.5). Temperature inactivation of the isozymes was shown by preincubating the isozymes for 10 min at graded temperature of 070°C. Kinetic studies were also performed on the isozymes. Statistical analysis Student's t-test was used for analysis of the data, and a p value of
J. Exp.
Med., 1992,
Thymidine Adenomatous
168, 291-301
Kinase
Activity
in Familial
Polyposis
SHINOBUSAKAMOTO and RYOHEI OKAMOTO Department of Function Disorder Research, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113
SAKAMOTO, S. and OKAM0T0,R. Thymidine Kinase Activity in Familial Adenomatous Polyposis. Tohoku J. Exp. Med., 1992, 168(2), 291-301 Thymidine kinase (TK) activity of polyp tissue from patients with familial adenomatous polyposis (FAP) was measured and compared with that of normal colon, sporadic polyp and colorectal carcinoma tissues. Total TK activity in colonic carcinoma was 3-fold that of normal ; this increase seems attributable mainly to increased activity of cytosolic TK isozyme activity ; the colorectal TK isozymes were separated into two types, i.e., fetal type and adult type isozymes predominantly in, respectively, cytosolic and mitochondrial fractions, by DEAEcellulose column chromatography. However, FAP polyp samples from 15 patients showed an average elevation of only 1.8-fold over normal. Examined individually, only 5 of the 15 FAP samples showed significant elevations in total TK activity. Furthermore, TK isozy u analysis revealed variable patterns of the cytosolic isozyme activity being elevated in some cases (8 of 15) and remaining low in others. Thus FAP polyps seem to be a heterogenous population with respect to DNA replicative activity, and cytosolic TK isozyme activity may constitute a biochemical marker for the subsequent development of colorectal carcinoma in FAP. familial polyposis coli (FPC) ; tymidine kinase (TK) ; isozyme
Familial adenomatous polyposis (FAP) is a genetic disorder characterized by the development of numerous adenomatous polyps in the colon and rectum. This condition is associated with a high incidence of colorectal carcinoma. A genetic locus for FAP has been identified (Bodmer, et al. 1987) on chromosome 5q; however, the structure and function of this gene remain unknown. Furthermore, other genetic alterations have also been described in the multi-step process of colorectal tumorigenesis in FAP, including loss of heterozygosity on chromosomes 17p and 18q (Okamoto et al. 1988), alterations in the expression of genes such as c-myc and cfos (Sugio et al. 1988) and the suppression of genes such as the wild-type p53 gene (Finlay et al. 1989), and mutations leading to activation of ras genes (Sasaki et al. 1990). The precise roles of these genetic alterations in the sequence of events leading to neoplasia and malignant transformation, and the corresponding alterations in the biochemistry of tumor cells, have yet to be elucidated (Fearon and Vogelstein 1990). Addressfor reprints : 1-5-45 Yushima,Bunkyo-ku, Tokyo 113,Japan. 291
292
S. Sakamoto
and R. Okamoto
We have long been using isozymes of thymidine kinase (TK ; EC 2.7.1.21), a DNA-synthesizing enzyme (Kit 1978), to examine such issues in general ; using this tool, we have recently focused on FAP in particular. Though abnormal nucleotide metabolism has previously been observed and implicated in the development of colonic cancer (Dutrillaux and Mulevis 1986; Luccioni et al. 1988), little is known about nucleotide synthesis in FAP patients. In the present study, we report the changes we have observed in the activity of thymidine kinase and its isozymes in colorectal tumors from patients with colonic cancer, with sporadic adenomatous polyps, and with FAP. MATERIALS AND METHODS Patients and materials Samples of normal colonic mucosa and colonic carcinoma were obtained from 60 patients [29 males, average age 65.2±2.0 years (range : 51-89 years), and 31 females, average age 63.1±2.3 years (range : 35-89 years)] who underwent surgical resection at the 2nd Department of Surgery, Tokyo Medical and Dental University, Japan. Upon resection, the tissues were immediately placed on ice and transferred from the operating room to the laboratory within 30 min. While on ice, the neoplastic lesions were separated from normal mucosa, and both specimens were divided into two parts, one for histology and another for enzymatic assays. The latter samples were stored at -80°C. Sporadic adenomatous polyps were resected from 15 patients [7 males, 8 females ; average age 49.6± 4.4 years (range : 14-65 years)], and the tissue specimens were immediately placed on ice and divided as described above. FAP polyp tissues were also obtained from 15 patients [9 males, 6 females ; average age 36.7±2.8 years (range : 22-52)], who underwent surgical resection for FAP. The diagnosis was established prior to resection by family history, as well as physical, radiologic, and rectosigmoidoscopic examination, and confirmed at the time of operation. These samples were also processed as described above. Preparation of enzyme extract and isolation of TK isozymes A crude enzyme extract was first prepared by pulverizing the tissue specimens with an autopulverizer under liquid nitrogen, followed by homogenization at 0°C with 10 volumes of buffer containing 5 mM Tris-HC1(pH 7.5)/0.1 mM EDTA/1 mM mercaptoethanol/0.25 M sucrose, and then collecting the supernatant after centrifugation at 105,000 g for 1 hr at 4°C. This crude extract was then used for assay of total TK activity, as described below, or fractionated by adding saturated ammonium sulfate to 50% saturation and stirring for 1 hr, followed by centrifugation at 10,000 g for 20 min. The precipitate was then resuspended in buffer containing 5 mM Tris-HC1 (pH 7.5)/20% glycerol/1 mM MgC12,and subjected to overnight dialysis against the same buffer, followed by centrifugation of the dialysate at 10,000 g for 30 min. The resultant supernatant was loaded onto a DEAE-cellulose (DE-52, Whatman, Kent, UK) column (1.5 by 5.0 cm) that had been equilibrated with the same buffer, and eludet stepwise from the column with 6 ml volumes of the same buffer containing increasing concentrations of NaCI (0 M, 0.1 M, 0.2 M, and 0.3 M). Fractions of 1.0 ml were collected and used for assay of TK isozyme activity, described below. Assay of thymidine kinase (TK) activity As previously reported, TK activity was measured by the method of Taylor et al. (1972). An assay mixture consisting of 5 mM MgC12,10mM ATP, 2pM [6 3H] thymidine, and 0.1 M Tris-HC1 buffer (pH 7.5) in a total volume of 200 p1 was incubated with either the crude enzyme preparation (for determination of total TK activity), or with fractions eluted
TK Activity
in Familial
Adenomatous
Polyposis
293
from the DEAF-cellulose column (for determination of TK isozyme activities), for 15 min at 30°C, and boiled to stop the enzyme reaction for 3 min, followed by centrifugation at 10,000g for 10 min. A 100,u1aliquot of the supernatant was spotted onto DEAE-cellulose paper, which was then washed extensively with 1 mM ammonium formate and ethanol to remove all unphosphorylated substrate, and dried ; the radioactivity remaining on the paper was then measured in a liquid scintillation counter. Characterization of biochemicalproperties of colonic TX isozymes Differential inhibitory effect of pyrimidine nucleotides on the TK isozymes was demonstrated by including deoxythymidine triphosphate (dTTP) at a final concentration of 1.0 mM, or deoxycytidine triphosphate (dCTP) at final concentrations of 0.5 mM, 1.0 mM, and 5.0 mM, in the reaction mixture. The molecular weights of the colonic TK isozymes was estimated by HPLC with use of a TSK-G3000SW column (600 by 7.5 mm internal diameter, Toyo Soda, Tokyo). The column was eluted with 0.1 M phosphate buffer containing 0.1 MNaOl (pH 6.5) at a flow rate of 0.8 ml/min. The effects of pH on the isozyme activities was studied by varying the pH of the reaction buffer with 0.1 M Tris-HCl (pH 7.0-9.5). Temperature inactivation of the isozymes was shown by preincubating the isozymes for 10 min at graded temperature of 070°C. Kinetic studies were also performed on the isozymes. Statistical analysis Student's t-test was used for analysis of the data, and a p value of
