platelet derived endothelial cell growth factor in human gastric carcinoma

t

ELSEVIER

CANCER LETTERS

I Cancer Letters 108 (1996) 281-290

Tumor angiogenesis and expression of thymidine phosphorylase/ platelet derived endothelial cell growth factor in human gastric carcinoma N. Tanigawaa,*, H. Amaya”, M. Matsumuraa, Y. Katoha, A. Kitaokaa, T. Aotake”, T. Shimomatsuyaa, O.A. Rosenwasserb, M. Iki” ‘The Second Department of Surgery, Fukui Medical School, Matsuoka-Cho, Yoshida-Gun, Fukui, 910-11, Japan “Department of Immunology and Parasitology, Fukui Medical School, Matsuoka-Cho, Yoshida-Gun, Fukui, 91 O-l I, Japan ‘Department of Environmental Health, Fukui Medical School, Matsuoka-Cho, Yoshida-Gun, Fukui, 910-11, Japan

Received 2 August1996;revisionreceived17September 1996;accepted 18September 1996

Abstract To investigatethe relationshipbetweentumorangiogenesis and the expression of platelet-derived endothelial cell growth factor/thymidinephosphorylase (PD-ECGF/dThdPase) and betweenpatients’ survival and the expressionof PD-ECGF/ dThdPasein humangastriccarcinomatissues,we performedimmunohistochemical studieswith anti-PD-ECGF/dThdPase andant&CD34monoclonalantibodies.Out of 154gastriccarcinomatissuesamples,61 (40%) wereevaluatedasPD-ECGF/ dThdPase-positive. The expressionof PD-ECGF/dThdPase wassignificantlyassociated with the intratumoralmicrovessel counts(P < 0.0001)andthe incidenceof hematogenous metastasis (P <: 0.05).Intratumoralvesselcountsweresignificantly correlatedwith overall survival of 154patients(P < O.OOOOOl). Cox proportionalhazardsmodelshowedthat tumor vasculature wasan independentandstrongprognosticvariable.However,the prevalenceof tbe expressionof PD-ECGFdid not associate the overall survival.We suggestthat expressionof PD-ECGF/dThdPase playsa role in the promotionof angiogenesisin humangastriccarcinomas,without any definiteinfluenceon patient’sswival. Keywords:

Thymidinephosphorylase (dThdPase);Platelet-derivedendotbelialgrowthfactor (PD-ECGF);Microvesselcount;

Prognosis; Gastric carcinoma

1. Introduction

resection [3]. Thymidine phosphorylase (dThdPase), alsoknown asplatelet derived endothelial growth fac-

Solid tumors mustinduce a vascular stromato grow beyond a minimal size [ 1,2], and we previously demonstrated that the intensity of the angiogenic

tor (PD-ECGF), is an enzyme that has been implicated in tumor-associated angiogenesis [4]. dThdPase is expressed at a higher level in tumors than in neighboring normal tissue in a variety of malignancies, including gastric carcinomas [5-91. Cells transfected with the PD-ECGF/dThdPase gene conferred a higher angiogenic potential in a nude mouse model than

response correlates with the prognosis of patients with gastric carcinomas who underwent curative

-* Corresponding author.

0304-3835/96/$12.00 PII

SO304-3835(96)04482-5

0 1996 Elsevier Science Ireland Ltd. All rights reserved

the non-trans&ctant cells [ 101. Other workers have found that the expression of PD-ECGF/dThdPase shows a significant correlation with tumor angiogenesis in some human tumors [Il. 121. However. as far as we are aware. there is no report describing the possible association between expression of PDECGF/dThdPase and tumor angiagenesis in gastric carcinomas. To determine whether PD-ECGFI dThdPase plays a role in human gastric carcinomas. we examined its expression in carcinoma tissues by an immunohistochemical method using an anti-PDECGF/dThdPase monoclonal antibody. The expression of PD-ECGF/dThdPase in carcinoma cells was compared with various clinicopathological features of the carcinomas. including intratumoral microvessel density, and overall survival. We report here that expression of PD-ECGF/dThdPase in tumor cells correlates with tumor angiogenesis. but not with the prognosis of human gastric carcinomas.

2. Patients and methods 7.1. Gastr-ic, carcinomas A total of 154 primary gastric carcinomas, which had been resected in our department from October 1983 to December 1993, were used in this study. Patientshad received neither chemotherapynoTradiation therapy before surgery. Pathological diagnosis and classification of disease stages were decided according to the General Rules for Gastric Cancer Study in Surgery and Pathology in Japan [ 131.They were all adenocarcinomas. Of these patie~nts.20 ( 13%) had stage 1 disease,17 ( 11%) had stage2 disease,53 (34%) had stage3 disease,and 64 (42%j had stage4 disease.One hundredand three patients (67%) underwent curative resectionand the other 5 1 patients underwent non-curative resection. No surgical deaths were encountered in this series.Ninety-two patients (60%), lessthan 70 years old, were treated with tegaful (2-tetra-hydrofuryl)-5-fluorouracil) at 600 mgJday for 2 years after surgery. All patients were reviewed at 2 or 3 monthly intervals in the outpatient clinic. Those patients who failed to attend the clinic were traced by telephone. If the patient had died, the date and causes of death were recorded. Tumor specimenswere fixed in 10% buffered formalin and embeddedin paraffin. Histological features in tumors were assessed using

hematoxyhn and eosin-stainedsectionsand included differentiation grade and depth of penetration, as well asblood vesseland lymphatic invasion. standard

-7.-. 7 PD-ECGFidThdPase monocolonui untihod\ The anti-PD-ECGF/dThdPase monoclonal ant!body. usedin this study?had been preparedfrom the human colon-cancer xenograft HC116 in the Department of Oncology, Nippon Roche ResearchCenter (Kamakura, Japan). In order to confirm the specificity of the anti-PD-ECGF/dThdPase monoclonal antibody, a lung cancer cell line PC9 transfected with PD-ECGF cDNA in our laboratory was used. PDECGF cDNA waskindly provided by Dr. S. Akiyama (Institute for Cancer Research, Kagoshima Univer ~ sity, Japan)with permissionof Dr. K. Miyazono tLudwig Institute for CancerResearch,Uppsala, Sweden). The transfected cells. PC9-DPE2, not only yielded approximately 50 times higher levels of dThdPase activity than the parental PC9 cells, but also showed strong immunostaining with a mouseanti-PD-ECGF! tlThdPasemonoclonal antibody, while PC9 cells were only faintly stained[ 141.For an immunoblot analysis. PC9-DPE2 cells and parental PC9 cells, as well as normal human liver tissue as a control, were lysed in buffer containing -50 mM Tris-HCl (pH ‘8.8), 10% glycerol, 1% sodium dodecyl sulfate (SDS), 1% 2-mercaptoethanol and 0.0005% bromphenol blue. The lysates were electrophoresedthrough 10% SDS/polyacrylamide gels (20 &lane). Proteins in the gel were electroblotted on to a nylon membrane(pore size 0.45 PM; FunakashXo. Ltd., Tokyo, Japan).The nylon membranewas blocked with 3% skimmedmilk in phosphatebuffered saline(PBS), andprobed with 2 @g/mlof the mouseanti-PD-ECGF/dThdPase monoclonal antibody. Biotinylated goat anti-mouseimmunoglobulin (Dako Japan, Kyoto, Japan)was used as a hecondary antibody. After the membrane had been incubated with 5treptavidin (Dako Japan. Kyoto. Japan), 10 ml of PBS containing 5 mg 3,3’-diamino. benzidine and 3 ~1 of 30% hydrogen peroxide were usedas substrate. 3.3. lmmunohistochemistry Expressionof PD-ECGF/dThdPase, CD34 and van Willebrand factor (vWF) were assessed by an indirect

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immunoperoxidase method. Four to 6 pm thick sections, mounted on glass slides, were dewaxed in xylene. dehydrated in ethanol. When staining for PD-ECGF/dThdPase, the slides were heated twice in an oven at 70°C for 5 min. They were then incubated in 3% hydrogen peroxide for 5 min. For quantitating tumor vascularization, anti-CD34 monoclonal antibody (QB-END/lo, Novocastra Lab., Newcastle, UK) and anti-vWF monoclonal antibody (F8/86, DAK0 A/S, Denmark) were used for staining vascular endothelial cells. After incubation in 10% normal bovine serum for 5 min, each slide was incubated overnight with the relevant monoclonal antibody at a 1:25 dilution. Biotinylated goat anti-mouse immunoglobulin and anti-rabbit immunoglobulin (Dako LSAB kit, Dako Japan, Kyoto, Japan) were used as secondary antibodies. Peroxidase-conjugated avidin (Dako Japan) was used at a dilution of 1:500. The slides were counterstained with hematoxylin and examined under low power (40 x ) to identify the region of highest PD-ECGF/dThdPase staining and that of highest vessel density. The expression of PDECGF/dThdPase was characterized as negative or positive, according to staining intensity, under a 200 x field. For vessel counting, a 200 x field in each of the five most vascular areas was counted, and the average counts were recorded. A vessel lumen was not required for identification of a microvessel, Normal mouse immunoglobulin G (IgG) diluted to an equivalent protein concentration was used as a control in place of the primary antibody for both CD34 and PD-ECGF/dThdPase stainings. Characterization of PD-ECGF/dThdPase expression and vessel counting were performed by two investigators, M.M. and H.A., who had no knowledge of the other clinicopathological features and clinical outcomes, using a double-headed light microscope simultaneously. 2.4. Statistical analysis The clinical characteristics of the patients were compared to the expression of PD-ECGF/dThdPase in the tumor cells and checked by the Mann-Whitney and chisquare tests. The survival curves were plotted according to the Kaplan-Meier method and their statistical differences were analyzed by the generalized Wilcoxon test. The influence of tumor vascularity on

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survival was assessed by the Cox proportional hazards model. For all statistical analyses, the SAS System for personal computers (SAS Institute Inc., Cary, NC) was used with significance having been defined as P < 0.05.

3. Results 3.1. Patients outcome Forty-nine of 154 patients (32%) have survived with a median survival of 51 months, ranging from 24 to 141 months, and the remaining 105 (68%) died between 2 and 77 months (median 12 months) after surgery. Peritoneal metastasis occurred in 7 1 patients (46%); 34 metastases were found at surgery and 37 developed after surgery. Hematogenous metastasis occurred in 33 patients (21%); 12 liver metastases were found at surgery and 21 developed after surgery (ten, liver; five, lung; two, bone; two, bone marrow; and two, brain). Both peritoneal and hematogenous metastases occurred in eight patients (5%); six liver metastases were found at surgery and two developed after surgery (one liver and one lung). Lymph node metastases were found in 120 patients (78%) at surgery. Seven of 49 survivors had metastatic diseases. 3.2. Western blot analysis of an anti-PD-ECGFI dThdPase antibody In the immunoblot analysis using the anti-human PD-ECGF/dThdPase monoclonal antibody, we detected a single protein band of 55 kDa in the PC9DPE2 cell lysate, which was identical to the band detected in normal liver tissue (Fig. I). The presence of a single band of the correct size in the PC9-DPE2 cell lysate and its absence in the parental PC9 cell lysate and strong immunostaining of the PC9-DPE2 cells with a mouse anti-PD-ECGF/dThdPase monoclonal antibody indicated that the anti-PD-ECGF/ dThdPase monoclonal antibody used in this study is specific for human dThdPase. 3.3. PD-ECGFldThdPase carcinomas

expression in gastric

The staining of PD-ECGF/d’IhdPase was mainly identified in the cytoplasm. In some tumors, expres-

1

2 3

107 -.

w-

52-

+

36 --27 --19

--

Fig. I. Characterization of the ant]-PD-ECGFidThdPasc monoclonnl antibody. Twenty kg of tissue lysate from normal liver tissue was used for lane 1, 20 pg of PC9-DPE2 celi lysate for lane 2. and 20 pg of PC9 cell lysate for lane 3. A 55 kDa protein band wax detected in lysates from liver tissue and PC9-DPE2 cells, while it was absent in PC9 cells lysates protein

sion was restricted to the neoplastic element and in others. non-carcinomatous mucosal cells or stromai cells were also weakly stained. In latter cases, the staining was defined as positive when expression of PD-ECGF/dThdPase was more pronounced in carcinoma cells than neighboring tissue. No specific staining was seen when control IgG was substituted for the primary antibody. Marked heterogeneity was observed in the distribution of tumor cells stained with PD-ECGF/dThdPase (Fig. 2). Sixty-one of 1.54 gastric carcinomas (40%) were positive for PDECGF/dThdPase expression.

Vascular endothelial ceils were stained with antiCD34 and anti-vWF antibodies, whereas no specific staining was seen when normal IgG was used. Intratumoral microvessel counts for the 154 tumors ranged from 19 to 440 with a mean value plus or minus standard deviation (MSD) of 157 + 77 (median 15.5) fat CD34 staining and from 4 to 272 with 39 + 34 MSD (median 31) for vWF staining. Staining with the antibody against vWF was variable, resulting in approximately 2S% of the total endothelium being stained with anti-CD34 antibody. .Although correlation of the microvessel counts for the two antibodies was significant (I’ = 0.503. P < 0.0011. the data presented

below were taken from anti-CD34 staining only, since the CD34 antibody gave both more reliable and specific staining of microvessels. The mean intratumoral microvessel count was 109 & 53 MSD for 49 survi *t’ors and 180 -i: 77 MSD for 105 died patients i/’ i 0.0001 by Mann-Whitney U-test). A median value of I.55 was taken as the cutoff point for discrimination of the lS4 patients into two subgroups: 78 patients with hypovascular tumors and 76 with hyperc,ascular tumors. Among the clinicopathological vari;thles examined. age. sex. Borrmann classificat.ion. tumor histology. depth of penetration. lymphatic invahion. venous vessel invasion, adjuvant chemotherapy. peritoneal metastasis. and lymph node metastasis were equally distributed among these two subgroups. However. hematogenous metastasis, advanced stage of disease and non-curative resection were more frequent in the hypervascular group (Table 1).

l‘he clinicopathological data from 6 1 patients whose tumors were positive for PD-ECGF/dThdPase expression and 93 patients whose tumors were negarive for PD-ECGF/dThdPase expression were compared. Except for microvessel counts and hematogenous metastasis, there was no statistical car.relation between PD-ECGF/dThdPase expression and the clinicopathological variables examined (Table 2). Expression of PD-ECGF/dThdPase correlated significantly with microvessel counts (P < 0.0005 by chi. .quare test ). The mean vessel counts were 1X2 Z!ZX0 ‘MSD for PD-ECGF/dThdPase-positive tumors and 142 k 72 MSD for PD-ECGF/dThdPase-negative mmors tf’ ,: 0.005 by Mann-Whitney test). The number ot tumors which were positive for PDECGF/dThdPase expression was plotted against the rnicrovessel count divided into progressive 100 vessel increments. The histograms showed that the prevalencc of PD-ECGF/dThdPase-positive tumors Increased as the vessel count within the tumor increased (Fig. 3).

We found, using the Cox model, that microvessel

N. Tanigawa

Fig. 2. lmmunohistochemical cells (orIgina magnification

staining x 200).

et al. i Cancer

for PD.ECGF/dThdPase

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counts correlated significantly with the overall survival of the patients even when evaluated as continuous variables (P < 0.0001, odds ratio 1.008 with 95% confidence limits ranged between 1.005 and 1.010). The odds of death increased with the number of intratumoral microvessel counts. The survival rates of the hypovascular and hypervascular groups were calculated using the Kaplan-Meier method. There was a statistically significant difference in the survival rate in favor of the hypovascular group. The 5 year survival rate was 47.9. + 6.4% mean plus or minus standard error (MSE) for the hypovascular group and 9.7 f 3.6% MSE for the hypervascular group (P < 0.00001). The Cox proportional hazards model identified the microvessel count as the significant prognostic variable which was independent from other clinicopathological variables, including stages

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tissues of the stomach.

285

There is a strong staining

in the tumor

of disease and operative curability (Table 3). On the contrary, the overall survival rates for 61 patients with PD-ECGF/dThdPase-positive tumors and 93 with PDECGF/dThdPase-negative tumors were not significantly different. When examined within each subgroup of tumor vascularity and PD-ECGF/dThdPase positivity, the 5 year survival rates were as follows: for 41 patients with hypervascular and PD-ECGF/ dThdPase-positive tumors 16.0 + 5.9% MSE, for 35 patients with hypervascular and PD-ECGF/dThdPase negative tumors 6.2 f 4.2% MSE, for 20 patients with hypovascular and PD-ECGF/dThdPase-positive tumors 55.0 4 11.1% MSE, and for 58 patients with hypovascular and PD-ECGF/dThdPase negative tumors 43.5 + 8.1% MSE. In both hypovascular or hypervascular group, patients with PD-ECGFI dThdPase-positive tumors tended to survive longer

rable

Table 2

1

Comparison hypovascular

of clinicopathological features between patients tumors and those with hypovascukr tumors

Variable

Age 5 b5 years >65 years Sex Male Female Borrmann classification I. 2 3. 4, unctassified Tumor histology Well differentiated Poorly differentiated Depth of penetration” tl. t2 13. t4 L.ymphatic invasionh IyO, lyl IyZ, ly3 Venous vesel invasionC \,o. “I VI. v.3 Adjuvant chemotherap? Negative Positive Peritoneal metabtaCI\ Ycgativc Positive Lymph node metastasis Negative Pohitive Hematogenouz, metastasis Negative Positive Stage of disease Sklge I stage 2 Stage 3 Siage 4 Surgical curability Curative Non-curative

Hypovascular (n = 78)

HypervascuLarP (n = 76)

with

value

Comparison of clinicopatholq$cal feaEures between tive and positive for PD-ECGIJ/d’%&~ expression

Variable

Expression ( - t (n = 93)

tumors

nega.

expression P value ( t ) (n = 611

AS 40 (51) 38 (49) 49 (63 i 29 (37)

30 (38) 4X (62) 41 (53) 37 (47)

35 (45) 43 (55) 24 (311 54 (69)

56 (72) 22 (281

4X (62i 30 13X) 4-l I%, 34 (341

5 bS year?, ->65 years sex Male Female Borrmann classification I. 2 3. 4, unclassified Tumor histology welt differentiated Poorly differentiated Depth of penetration” tl, t2 t3, t4 Lymphatic invasion” iyo. lyl ly2, ly3 Venous vessel invasion’ \,(I. c I v2. v3 Adjuvant chemotherapy Qgative Positive Peritoneal metastuhl\ Nsgal iv? f+>sitivr Lymph nod{: metaqtasl, \;egati\.t. r’I~‘,lllvc I lematogcnou\ met3staG

46 (49) 47 (511 60 (65) 33 (35) 31 (33, 62 (67) 42 (45) 51 W-7) 3s (3X11 58 (62) 24 (?b) 64 (74)

h.? (40) 31 (XI

Negative

22 (2X) 56 (721

70 VW) x (101 I? II 31 23

(171 (1-l) (40, (29)

59 (76) 19 (24)

KS, no significant difference. “I I. penetrates to submucosa: tY+ to suhserosa: t3, serosa (exposed). t4. infiltrating the neighboring tissue.

Posiliw Stage of disease Siage L Svage 2 stage :i S1agr 1 Surgical curabilit? Curativc Non-curative Microvchscl count\ . I55 .f 155

b3 tbRI 30 (3,)

NS. no signihcant-difference. “t I, penetrates to submucosa; t?. to subserosa; t-1. mfiltrating the neighboring tissue. hlyO--I~t3, grade of lymphatic vessel mvas~. ‘vOh3. grade of venous vessel invasion.

0, serosa (exposed):

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Table 3 Cox proportional

hazards

model for overall

survival

Variables

P value

Relative risk

95% confidence limits

Vessel counts (Cl55 or more) Stage of disease (1, 2, 3, and 4) Surgical curability (curative or non-curative)

0.0001

2.543

1.671, 3.869

0.0001

1.925

1.490, 2.485

0.9457

1.023

0.534,

1.959

than those with PD-ECGF/dThdPase-negative tumors, but again the differences were not statistically significant. 4. Discussion Recent studies have demonstrated that dThdPase is almost identical to PD-ECGF [4,15-171. Our immunohistochemical analysis of human gastric carcinomas demonstrated that 40% of tumors expressed PD-ECGF/dThdPase. This result appears to be compatible with the data reported by Toi et al. who found, using the same monoclonal antibody, 39% of its expression in invasive ductal breast carcinomas [ 111. Others have also reported that the PD-ECGF/ dThdPase expression occurs more frequently in human colorectal carcinomas than in adenomas or normal mucosa [ 121. Meanwhile, several biochemical studies have indicated that the level of dThdPase activity is elevated in many types of malignant tumors including gastric, colorectal, and breast carcinomas, when compared to normal tissues [5]-[9,12]. Therefore, the immunohistochemical results using the antiPD-ECGF/dThdPase monoclonal antibody appear to be concordant with the data of dThdPase activity. dThdPase, an enzyme involved in pyrimidine nucleotide metabolism, has recently been shown to have angiogenic activity [4,18-201. However, the mechanism of angiogenesis by dThdPase has not yet been fully elucidated. Haraguchi et al. recently indicated that dThdPase, through the degradation products of thymidine, stimulates the chemotaxis of endothelial and possibly other cells and thus indirectly induces angiogenesis [21]. The current study of human gastric carcinomas indicates that the expression of PD-ECGF/dThdPase correlates significantly

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with intratumoral microvessel counts and that the prevalence of PD-ECGF/dThdPase-positive tumors increases as the intratumoral vessel count increases. This is consistent with the findings of other studies using invasive breast and colorectal carcinomas [11,12]. Many studies have demonstrated that the intratumoral vessel count was the significant predictor of overall survival in a variety of malignancies [22281. Our previous study extended these findings to gastric carcinomas which had undergone curative resection and demonstrated that intratumoral vascularization correlated significantly with overall survival as well as hematogenous metastasis [3]. In addition, consistent with other reports [27,29], we confirmed that the CD34 antibody gave both more reliable and specific staining of microvessels than anti-von Willebrand Factor antibody. Therefore, the data of vessel counts were taken from the anti-CD34 staining. The current study group of 154 patients contained not only patients who had received curative resection but also those who had received non-curative resection, confirmed all the above findings. The Cox model indicated that intratumoral vessel counts correlated significantly with the overall survival of the patients whenever evaluated as continuous variables

IKi31

1Oll-101) Micro\wvzl

h/IO

2IHl-3ll1l count<

Fig. 3. Relationship between PD-ECGF/dThdPase expression and microvessel counts. The prevalence of the positivity of PD-ECGF/ dThdPase expression increased as the intratumoral vessel counts increased.

or divided into the hypovascular and hypervascular groups. Of interest, however, is that the expression of PDECGF/dThdPase was not found to correlate with the overall survival rate, although PD-ECGF/dThdPase expression was associated with higher vessel counts. The survival analyses in the subgroups divided into tumor vascularity and staining for PD-ECGFI dThdPase indicated that patients with PD-ECGF/ dThdPase-positive tumors tended to survive longer than those with PD-ECGF/dThdPase-negative tumors in either hypervascular or hypovascular group. Heldin et al. [30] indicated that PD-ECGF/dThdPase was more frequently expressed in more differentiated types of human non-small cell lung cancer cell lines. Although we found no significant relationship between PD-ECGFldThdPase expression and histological phenotype. the aforementioned survival results may indicate the relevance of PD-ECGF/dThdPase expression to differentiation in gastric carcinoma cells. Recently, the various other peptide growth factors. such as vascular endothelial growth factor (VEGF) 1311,basic fibroblast growth factor (bFGF) [32], hepatocyte growth factor (HGF) 1331, and transforming growth factor OI-(TGF~) 1341 have been found to stimulate proliferation and motility of endothelial cells, thus inducing new blood vessel formation. In addition, several factors that down-regulate or inhibit angiogenesis have been identified. They includes angiostatin [ 351, interferon-cu-(IFNa) 1361. thrombospondin-1 (TSPl) [37] and platelet factor 4 (PF4) [38]. and some have already been incorporated into clinical trids 1391. As tumor angiogenesis is a complex multi-step process controlled by such various factors. it is unIikely that the angiogenic phenotype of human gastric carcinomas can be explained by the expression of PD-ECGF/dThdPase alone. dThdPase has received considerable attention as a moduIator of chemotherapeutic drugs, Doxifluridine and tegaful are converted to S-fluorouracil (5FUra) and 5FUra is converted to fluoro-deoxyuridine monophosphate by dThdPase in tissue. Haraguchi et al. attained enhancement of sensitivity to doxifluridine and tegaful by transfection of PD-ECGF cDNA into KB cells 1401. Recently. we found ~that PD-ECGF cDNA-transfected PC9 cells were more sensitive not only to doxifluridine and tegaful but also to 5FUra

than untransfected PC9 cells [ 141. Thus, with respect to tumor angiogenesis, the antitumor activities of doxifluridine, tegaful and SFUra, being widely used in the treatment of various kinds of tumors. may be important in the selective inhibition of those tumor cells expressingPD-ECGF/dThdPase. The mechanismsthat control angiogenesisin different tumors are likely to be highly diverse [41]. Abundant messagefor VEGF was found in human colorectal cancer cells and gastric carcinomas using immunohistochemistryand in situ hybridization [42 //. Takahashiet al. recently demonstrateda high correlation between vessel counts and VEGF expression as well as expression of flk-1, a receptor of VEGF. in human colon cancer specimens[43]. Other researchers have shown that systemic administrdtioll of a11 antibody to VEGF inhibits the in vivo. but not iri vitro, growth of human colon xenografts and leads IO a marked reduction of liver metastasis[44]. Since we consider that these findings on human colon carcinomas can be extended to gastric carcinomas. we have recently conducted experiments to clarify whether the YEGF/flk system may play a role in the angiogenesis of human gastric carcinomas [45]. Ascertainment of factors which are highly relevant to tumor angiogenesis,based on the data of tumur vasculature as well as clinical outcomes like this study, may have an influence on future development of new treatment modalities which may inhibit tumor growth and metastatic formation by inhibiting angiogenesis. References Folkman. J. I IYXh) How is blood vessel growth regulated in normal and neoplastic tissue?, (G.H.A. Clowes Memorial Award Lecture). Cancer Res., 46, 467-471. Falkman. J. (1990) What is the evidence that tumors arc angiogenesis dependent?, J. Natl. Cancer inst.. 82, 4-6. Tanigawa, N.% Amaya, H.. Matstmiura. M., Shimomatsuya T.. Horiuchi. T.. Muraoka. R. and Iki, M. (1996) Extent of tumor vascularization correlates with prognosis and hematogenous metastasis in gastric carcinomas, Cancer Res.. 56. 267 1- 2616. Furukawa. T., Yoshimura, A.. Sunutawa, T.. Haraguchi, M.. Akiyama, S.. Fukui, K., Ishizawa, M. and Yamada. K.( 19921 Angiogenic factor. Nature (London). 356. 66R. Zimmerman. M. and Seidenber. J.(1964) Deoxyribosyl transfer. J. Riol. them., 239, 26%.2621, Pauly, J.L.., Paolini. N.S.. Ebarb, R.L. and Germain, M.J.

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(1978) Elevated thymidine phosphorylase activity in the plasma and ascites fluids of tumor-bearing animals, Proc. Sot. Exp. Biol. Med., 157, 262-267. Miwa, M., Nishimura, J., Kamiyama, T. and Ishitsuka, H. (1987) Conversion of 5’-deoxyfluorouridine to 5-FU by pyrimidine nucleoside phosphorylases in normal and tumor tissues from rodent bearing tumors and cancer patients, Jpn. J. Cancer Chemother., 14, 2924-2929. Yoshimura, A., Kuwazuru, Y., Furukawa, T., Yoshida, H., Yamada, K. and Akiyama, S. (1990) Purification and tissue distribution of human thymidine phosphorylase: high expressoon in lymphocytes, reticulocytes and tumors, Biochim. Biophys. Acta, 1034, 107-113. Reynolds, K., Farzaneh, F., Collins, W.P., Capbell, S., Boume, T.H., Lawton, F., Moghaddam, A., Harris, A.H. and Bicknell, R. (1994) Association of ovarian malignancy with expression of platelet-derived endotbelial cell growth factor, J. Natl. Cancer Inst., 86, 1234-1238. Ishikawa, F., Miyazono, K., Hellman, U., Drexler, H., Wemstedt, C., Hagiwara, K., Usuki, K., Takaku, F., Risaw, W. and Heldin, C.H. (1989) Identification of angiogenic activity and the cloning and expression of platelete-derived endothelial-cell growth factor, Nature (London), 338, 557562. Toi, M., Hoshina, S., Taniguchi, T., Yamamoto, Y., Ishitsuka, H. and Tominaga, T. (1995) Expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human breast cancer, Int. J. Cancer, 64, 79-82. Takebayashi, Y., Yamada, K., Maruyama, I., Fujii, R., Akiyama, S. and Aikou, T. (1995) The expression of thymidine phosphorylase and thrombomodulin in human colorectal carcinomas, Cancer Lett., 92, l-7. Japanese Research Society for Gastric Cancer (1993) The General Rules for Gastric Cancer Study, 12th edn. Kanehara Public Co., Tokyo. Kate, Y., Matsukawa, S., Muraoka, R. and Tanigawa, N. (1’396) Enhancement of drug sensitivity and a bystander effect in PC-9 cells transfected with a platelet derivedendothelial cell growth factor/thymidine phosphorylase cDNA. Br. J. Cancer, in press. Usuki, K., Saras, J., Waltenberger, J., Miyazono, K., Pierce, Cr., Thomason, A. and Heldin, C.-H. (1992) Platelet-derived endothelial cell growth factor has thymidine phosphorylase acnvity, B&hem. Biophys. Res. Commun., 184, 1311-1316. Moghaddam. A. and Bicknell, R. (1992) Expression of platelet-derived endothelial cell growth factor in Escherichia coli and confirmation of its thymidine phosphorylase activity, Biochemistry, 31, 12141-12146. Sumizawa. T., Furukawa, T., Haraguchi, M., Yoshimura, A., Takeyasu, A., Ishizawa, M., Yamada, Y. and Akiyama, S. (1993) Thymidine phosphorylase activity associated with platelct-derived endothelial cell growth factor, J. B&hem., 114, 9-14. Finnis, C., Dodsworth, N., Pollitt, C.E., Carr, G. and Sleep, D. (1993) Thymidine phosphorylase activity of platelet-derived endothelial cell growth factor is responsible for endothelial cell mitogenicity, Eur. J. B&hem., 212, 201-210.

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[I91 Moghaddam, A., Zhang, H.-T., Fan, T.-P.D., Hu, D.-E.H., Lees, V.C., Turley, H., Fox, S.B., Gatter, K.C., Harris, A.L.

[201

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