J. Cancer Res. Clin. Oncol. 95, 273-280 (1979)

Journa~ of

Cancer Research and

Clinical Oncology �9 Springer-Verlag 1979

Incorporation Studies of Nucleic Acid Precursors in Gastric Cancer

An Attempt in Individualized Chemotherapy

P. Schlag 1, J. Veser 2, G. Geier 3, D. Breitig 4, and P. Merkle 1

1 Abteilung ffir Allgemeine Chirurgie (Leiter: Prof. Dr. Ch. Herfarth) des Departments fiir Chirurgie, Steinh6velstr. 9, D-7900 Ulm,

2 Abteilung ffir Biochemie I (Leiter: Prof. Dr. H. Thomas), 3 Department fi_ir Gyn~ikologie und Geburtshilfe (Leiter: Prof. Dr. K. Kn6rr, Prof. Dr. H. Lauritzen), 4 Zentraler Laborbereich fiir Isotopenanwendung (Leiter: Dr. D. Breitig)

der Universit~[t Ulm, Federal Republic of Germany

Inkorporationsstudien der Nucleins~iurepr~ikursoren bei Magenkrebs Ein chemotherapeutischer Versuch

Zusammenfassung. Durch Messung der Einbaurate radioaktiv markierter Nucleins~iurepr~ikursoren in die DNA bzw. RNA bei Magenkarzinom-Zell- suspension ergaben sich Hinweise auf eine unterschiedliche Proliferations- aktivit~it der Tumoren. Der Markierungsindex korrelierte im allgemeinen mit dem zytologischen Differenzierungsgrad des Karzinoms. Mit Entdifferenzierung der Tumoren stieg die Proliferationsrate an. Hieraus ergibt sich bei bekannter proliferationsabh~ingiger Wirkung der meisten Zytostatika eine weitgehende Zytostatika-Resistenz der hochdifferenzierten Magenkarzinome. Die Nuclein- s~iuresynthese proliferationsaktiver Tumoren konnte nur teilweise durch die getesteten Zytostatika (5-Fluoro-Uracil, Adriamycin) gehemmt werden. Kar- zinome mit Stoffwechselkompensationsm6glichkeiten gegenfiber dem Wir- kungsmechanismus der Zytostatika erwiesen sich biochemisch resistent. Eine Aussage iiber zytostatikasensible Tumorenist neben den sich hierbei ergebenden methodischen Problemen auch aufgrund nicht erfaf~ter Patient-bedingter Resistenzursachen anhand yon Incorporationsstudien problematisch.

Schliisselwiirter: Magencarcinom Nukleins~iure Synthese - Chemotherapie

Summary. Measurements of the rate of incorporation of radioactively labeled nucleic acid precursors into the DNA and RNA of gastric carcinoma cell suspensions indicated variable rates of proliferation for the tumors. The rate of incorporation generally correlates to the cytological level of differentiation of the carcinoma. Reduced differentiation of the tumors showed a corresponding increase in the rate of proliferation. Knowing the proliferation-dependent effect of most cytostatica, this results in a resistance to cytostatica of highly differentiated gastric cancers. The nucleic acid synthesis of proliferatively active tumors could only be partially inhibited by the cytostatica tested (5-fluorouracil, adriamycin). Carcinomas with metabolic possibility for compensation of the active mechanism of the cytostatica were biochemically resistant. Due to the

Offprint requests to: Dr. P. Schlag (address see above)

0171-5216/79/0095/0273/$1.60

274 P. Schlag et al.

resul t ing me thod ica l p rob lems and unaccoun tab le pa t i en t -dependen t causes of resistance, a conclusive s ta tement a b o u t cy tos ta t ica-sens i t ive tumors is difficult to m a k e in i nco rpo ra t i on studies.

Key words: Gas t r i c cancer - Nucle ic acid synthesis - C h e m o t h e r a p y - Predic t ive test.

A p a r t f rom the stage of the disease and its level of differentiat ion, the the rapy and prognos is of gast r ic cancer depend on the prol i fera t ive tendencies of the t u m o r (Bri t ton et al., 1975; Hawley et al., 1970; Ming, Si-Chung, 1977). The success of a cy tos ta t ic ad juvan t t he rapy is, a m o n g o ther things, a funct ion of the kinet ics of the t u m o r cell (Valer iote and van Put ten, 1975). I t is very difficult to influence the course of gast r ic cancer with cytostat ica . Of the few substances effective agains t this tumor , 5-f luorouraci l , m i tomyc in C, and der ivat ives of n i t ro sou rea are the mos t powerful (Moer te l et al., 1976). The effectiveness of a d r i a myc in agains t gas t r ic cancer is no t yet sufficiently d e m o n s t r a t e d (Moerte l , 1978). The response ra te to s ingle-drug the rapy with a p roven cytos ta t ic agent is quo ted at 20%. Since only a smal l percentage of gast r ic ca rc inomas responds to cy tos ta t ic t he rapy at all, i n fo rmat ion concern ing the sensi t ivi ty or res is tance of the t u m o r to cy tos ta t ica would be ext remely va luab le before beginning t rea tment . In this way, pa r t of the pat ients could be spa red a useless therapy, which is in some cases coup led with grave side effects. This s tudy was thus des igned to ana lyze the pro l i fe ra t ion behav io r of gast r ic ca rc inoma in vi t ro by de te rmin ing its ra te of nucleic ac id synthesis. A further po in t of interest was the influence of cy tos ta t ica on the me tabo l i sm of nucleic acids, wi th a view to future p lann ing of ind iv idua l ized chemothe rapy .

Methods

The investigations were carried out in modifcation of a method specified by Kummer (1971) and Volta (1970). The surgically obtained tumor material was transported to the laboratory in a sterile plastic container with Hank's solution at room temperature and immediately prepared. Fat and muscle tissue, blood coagnla, and necroses were removed and the tumor was mechanically cut to pieces in plastic petri-dishes under sterile conditions. The tissue particles thus obtained were again immersed in Hank's solution and cells isolated by means of a sharp-edged glass pipette. After filtering the cell suspension through a gauze (pore size 200 gin), the cells were centrifuged (10 rain, 200 g) and resuspended in the medium of incubation (nutrient medium according to Limburg, mixed with Cephalotin, 100 y/ml). The cell suspension was standardized for 10%cells/ml nutrient solution, distributed among test-tubes and pre-incubated in a water bath shaken at 37 ~ for 60 rain with and without cytostatica. The cytostatica used were 5-fluorouracil (10 .2 mg/ml) in a concentration ten times their therapeutic equivalent. Sixty and 90 min after marking it with methyl-3H-thymidine (0.1 mCi/ml, specific activity 20 Ci/mM), 5,6-3H-uridine (0.1 mCi/ml, specific activity 43 Ci/mM) or 6-3H-desoxyuridine (0.1 mCi/mt, specific activity 15 Ci/mM), 100 lal each of the cell suspension was taken from the test tubes. The cells were treated with 5% ice-cold TCA. The acid-insoluble precipitate was suctioned over Whatman roundfilters and washed with ether/ethanol (1 : 1) to remove the neutral lipids. The dried filters were dissolved in 1 ml Soluene (Prod. Packard) and the radio- activity was measured in a fluid-scintilloscope after the addition of 7 ml Instagel (Prod. Packard). The rate of incorporation of radioactively labeled nucleic acid precursors (cpm/100 gl cell suspension) under the influence of cytostatica was compared to the rate of incorporation exhibited by the cell suspension without cytostatica (control). Cell viability was determined by using trypan blue. A further parameter for cell viability was the continued incorporation of radioactively labeled substances throughout the time of incubation. The tumors were classified cytologically by means of smear preparations of the cell suspension stained according to Papanicolaou, by cell type, size of the nucleus,

NucLeic Acid Precursors in Gastric Cancer 275

Fig. la-c. Cytological smear of the cellular suspension of a highly differentiated a, medium differentiated b, and slightly differentiated e gastric carcinoma (Papanicolaou~ 160 x )

dissociation, and polymorphism. Depending on the cellular components most in evidence, carcinomas of high, medium, and slight differentiation were classified (Fig. la-c),

Results

Due to difficulties in the preparation, standardized suspensions of tumor cells could only be produced for 51 of 69 gastric carcinomas. Particularly in scirrhous

276 P. Schlag et al.

3H-Desoxyuridin- incorporation (Cpm)

1000-

500-

=.-:-.;:. =..

5()0 1000 3H-Uridin incorporation (Cpm)

Fig. 2. Relationship of 3H-uridine- and 3H-desoxyuridine incorporation in cellular suspensions of gastric carcinoma (n = 36)

3H-Thymidin- incorporation

(Cpm) 1000

5 0 0 -

O�9

O0

0 o~ �9 �9 �9

5C)0 1600 3H- Desoxy- Uridin incorporation (Cpm)

Fig. 3. Relationship of 3H-desoxyuridine- and 3H-thymidine-incorporation in cellular suspensions of gastric carcinoma (n = 19)

cancers, it was frequently impossible to isolate sufficient cells from the tissue. The rate of RNA synthesis of the tumor cells, measured by uridine incorporation, surpassed the rate of DNA synthesis, measured by the incorporation of thymidine and desoxyuridine. Uridine was not, or only to a slight extent, incorporated by 18 of a total of 51 tumor cell suspension (Cpm < 150). No incorporation of DNA- specific nucleic acid precursors took place in those tumors demonstrating a low

Nucleic Acid Precursors in Gastric Cancer 277

3H-Uridin- incorporation

(Cpm)

1000.

500.

ODD�9 � 9 1 4 9 1 4 9 1 4 9 1 4 9

highly differentiated

OO

8 ,lb

� 9 1 4 9 1 4 9

moderate differentiated

.tI OO

undifferentiated

Fig. 4. Relationship of cytological level of differentiation and nucleic acid synthesis (3H-uridine- incorporation) in cellular suspensions of gastric cancer (n = 51)

rate of RNA synthesis. On the other hand, increased uridine incorporation was coupled with an increased incorporation of desoxyuridine (Fig. 2). The incorpora- tion of thymidine, as a further DNA-specific label, was almost linear to the desoxyuridine incorporation (Fig. 3). In the gastric carcinomas investigated, DNA was thus synthesized by utilizing desoxyuridine ("de novo synthesis") as well as by the direct incorporation of thymidine ("salvage pathway").

Of the 51 cell suspensions, 14 were cytologically highly differentiated carcinomas, 18 were medium, and 18 only slightly differentiated. The summary proliferation of the malignomas, measured by the incorporation of radioactively labeled nucleic acid building-blocks, was variable. There was a correlation between the rate of proliferation and the level of differentiation of the tumor. Uridine incorporation was low in highly differentiated tumors, no DNA synthesis could generally be determined in these tumors during the time allowed by the experiment. Medium and particularly slightly differentiated tumors increasingly incorporated the respective RNA- and DNA-specific nucleic acid precursors as an expression of increased proliferation (Fig. 4).

The incorporation of radioactively labeled nucleic acid components could be partially inhibited by 5-fluorouracil and adriamycin. Cytostatic inhibition generally increased with the rate of uridine incorporation (Fig. 5). Given the working mechanism of the cytostatica tested carcinomas with little proliferative tendency remained largely unaffected. Adriamycin had the maximum inhibitory effect on the RNA-synthesis of the tumor cells. 5-Fluorouracil predominantly blocked desoxyuridine incorporation by inhibiting the synthesis of thymidilic acid. In this last case, however, there was a parallel increase of thymidine incorpora- tion in most tumor cell suspensions compared with the cell suspensions without cytostatica (Fig. 6).

278 P. Schlag et al.

Inhibitory- effect

% 100

5 0 -

�9 � 9 �9 �9

5 1(~00 aH-Uridin incorporation (Cpm)

Fig. 5. Inhibitory effect of adriamycin (10 .2 mg/ml) in relation to 3H-uridine-incorporation in cellular suspensions of gastric carcinoma (n = 38)

3H-Thymidin- incorporation ,

% + 5 0

+ 2 5 -

- 2 5 -

- 5 0 -

~

�9 �9 * 5~0 1()0 Inhibitory effect% a H - D e s o x y - U r i d i n - i n c o r p o r a t i o n

Fig. 6. Relationship of 3H-thymidine incorporation and the inhibitory effect of 5-fluorouracil (10 .2 mg/ml) on the 3H-desoxyuridine incorporation in cellular suspensions of gastric cancer (n = 20)

Discussion

It seems a proven fact, that the summary rate of proliferation of a tumor can be determined by measuring the rate of incorporation of radioactively labeled nucleic acid precursors (Seeber and Schmidt, 1977). A part of the gastric carcinomas revealed no or only slight tendency to proliferation. The question of how much

Nucleic Acid Precursors in Gastric Cancer 279

this depends on the large proportion of dormant cells (Go-fraction, non-dividing pool) or on the long duration of the cell cycle, must remain unanswered in this present study. It must further be considered, that the rate of incorporation of radioactively labeled nucleic acid building-blocks can also be influenced by the differences in the nucleotide-pool of the individual tumors (Seeber and Schmidt, 1977). Earlier studies were, on the other hand, able to demonstrate that the ratio of labeled and unlabeled nucleotides remains constant for the chosen time of incubation, when the incorporation of radioactively labeled nucleic acid com- ponents proceeds in a linear fashion (Kummer and Kraml, 1977).

The incorporation of individual marker substances was low in highly differentiated gastric carcinomas. The rate of proliferation increased with reduced differentiation of the tumor, though the two factors were not necessarily linked. Similar findings were already produced for breast cancer (Schlag et al., 1978). In keeping with the proliferation-dependent mechanism of function of most cytostatica, proliferatively inert tumors are for the most part resistant to cytostatica. In agreement with studies on experimental animals, cytostatic effects are generally better demonstrable, once the incorporation of individual nucleic acid precursors has accelerated (Volta et al., 1975). Depending on the point of attack and the mechanism of the cytostatica used, different steps in the synthesis of nucleic acids were inhibited. RNA synthesis and to some extent DNA synthesis was blocked by adriamycin (Chabner et al., 1977; Torrelli, 1977). 5-Fluorouracil, which inhibits the thymidilate synthetase once converted to 5-fluoro-desoxyuridine- monophosphate (Wilmanns, 1971), reduces the incorporation of desoxyuridine in the cell suspension. This effect was for the most part compensated by the majority of the tumors with an increased incorporation of thymidine. Tumors able to compensate this metabolically via the activation of thymidine kinase are only partially affected in their capacity for DNA synthesis by 5-fluorouracil (Kummer, 1971; Kummer and Kraml, 1977). It now also becomes obvious that incorporation measurements performed with only o n e precursor are not enough for the determination of the cytostatic effect on a tumor's nucleic acid synthesis, and may reflect a false state of affairs.

The inhibition of the nucleic acid metabolism of a tumor cell does not necessarily cause its death. Cytotoxic effects can be ascertained by methods directly analyzing the tumor cell's capacity for cell division (Roper and Drewinko, 1976). The "colony formation technique" has gained in importance for these analyses (Salmon et al., 1978). Incorporation studies with radioactively labeled nucleic acid precursors can thus be primarily restricted to furnishing information about tumors resistant to cytostatica (Hall, 1977; Seeber and Schmidt, 1977). Cancers with a reduced rate of proliferation or with the possibility for metabolic compensation of the working mechanism of a cytostaticum seem little suited for chemotherapy. A statement concerning a tumor's responsiveness to cytostatic therapy is also extremely difficult to make due to the patient's own resistance factors, such as variing metabolism, elimination, and inactivation of a drug. In vivo models with heterotransplantation of tumors in so-called nude mice can skirt these problems only to a certain degree (Bastert et al., 1977). In vitro methods on a cellular-biochemical level therefore still constitute the primary point of departure for an individually planned cytostatic therapy to date.

280 P. Schlag et al.

Conclusion

The investigations described permit a statement about the summary pro- liferative activity of a gastric carcinoma. Since most cytostatica are effective only for proliferating cell populations, tumors with a reduced rate of proliferation seem little suited for chemotherapy. Beyond that, cancers with a capacity of biochemically compensating the active mechanism of a cytostaticum on their metabolism can be determined. This additionally creates the possibility of ex- cluding ineffective cytostatica from a chemotherapeutic program.

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Received December 18, 1978/Accepted October 5, 1979