DNA content of tumours

Europ.3. CancerVol. 1, pp. 303-307. Pergamon Press 1965 Printed in Great Britain

D N A Content of Tumours

Cytophotometric Measurements WALTER SANDRITTER

Department of Pathology, ~ustus Liebig University, Giessen, Germany

measurements [3] demonstrated that the dry weight (interference microscopy) as well as the D N A and histone protein content showed values that deviated from the normal. Measurements on an oat cell carcinoma of the lung serve as an example. The dry weight of the cell nuclei amounted to 20-30 × 10-1~g (normal cell nuclei of the bronchial tree 4 0 - 5 0 × 10-1~g).

CYTOPHOTOMETRIC measurements of normal cells reveal a regular distribution of D N A values which corresponds to the chromosome number [l]. In human tumours, aneuploid D N A distribution patterns were often found that deviated from the normal [2]. Our own *Supported by the Deutsche Forschungemeinschaft.

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The histone protein content (fastgreen staining pH 8.2) as well as the DNA content corresponded to a hypodiploid DNA content (Fig. 1). Investigations on thirty h u m a n malignant tumours (Fig. 2) showed that most of the tumours have a DNA "stem line" lying between diploid and tetraploid or octoploid values. However, normal DNA values were also found. These measurements of smears do not indicate whether this atypical DNA distribution occurs only in the peripheral zone or as well in the centre of the carcinoma cell cord. Figure 3 shows measurements made from tissue sections. It is clearly seen that the outer and inner zones of the carcinoma cell cord had cells with an atypical DNA content and that the DNA stem line is expressed in the same way in both zones. perlpheric port Co Xll

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In experimental animal tumours in contrast to the situation in humans, a diploid DNA content is usually found [4]. What do early cancerous lesions show in the h u m a n being? Carcinoma in situ of the

exocerxvix exhibited, contrary to simple atypical epithelium (dysplasia) (Fig. 4), an aneuploid DNA stem line as seen in a total of eight cases. Figure 5 shows that this stem line was found in all layers of the carcinoma in situ. The stem line persisted throughout the different steps of the invasion (Fig. 6) up to the microcarcinoma [5]. This could also be found in a case of malignant lung adenomatosis (Fig. 7). The DNA distribution pattern represents a nearly triploid stem line, independent oflocalisation of tumour cell population (intra-alveolar, lymphangiosis carcinomatosa and lymph node metastasis) [6]. During cancerisation in vitro [7], usually after one year a new DNA stem line develops that persists unchanged over a long period of time (Fig. 8). After a stem line has developed the rat fibroblasts can be transplanted back into rats. These transplanted tumour cells show another stem line than the original cells. Upon retransplantation of the animal tumours in tissue culture the stem line changes often again. These investigations show that malignant tumours are often distinguished from normal

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Fig. 8. Scheme of the culture times of the cell stems, giving the DJVA measuring time (single cross-line) and the time the chromosomes were counted (double crossline). The symbols on the left side of the line stand for the chromosome counts and those on the right of the line for DJVA measurements. Round spot = diploid, square area = hypotetraploid, I~ = no stem line.

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cell populations by an aneuploid D N A stem line and a wide scattering of D N A values. However, tumours are also found having a regular D N A distribution. T h e question arises whether these are pseudodiploid tumours. Measurements of early cancerous lesions (e.g. carcinoma in situ) show that an abnormal D N A distribution pattern is already found with intra-epithelial atypical cell formations and that the D N A distribution remains the same during the invasive phase (realisation of the carcinoma). This leads to the conclusion that with regard to the DNA, the carcinoma in situ already represents a carcinoma and that the invasion depends on other factors than the atypical D N A distribution. T h e amount of DNA by itself presents interesting information. However, there is the question to what extent there are also quali-

tative differences in the nucleo-proteins of normal and tumour cells. Measurements made during the Feulgen hydrolysis (time curve) have shown that contrary to normal cells, mouse ascites tumour cells exhibit an atypical hydrolysis curve with an inflection at 9 min [8] (see Fig. 9). We believe that this atypical curve results from a differential acid sensitivity of the eu- and heterochromatin. When the hydrolysis time (HC1, p H 1 "2, 37 °) is extended, we also see in the case of normal cell nuclei a curve [9] with an inflection at about 20-22 hr (Fig. 10). Recent data suggest that in the first part of hydrolysis (up to 20 hr) the aldehyde groups of the euchromatin are set free, while the heterochromatin is less acid sensitive. Further investigations still need to be m a d e to resolve this relationship.

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D N A Content of Tumours RESUblE L'auteur t,bnne un bref aper~u de ses travaux sur la cytophotom~trie des tumeurs malignes, notamment ?t la phase prdcoce de l'affection. II rut possible de montrer que les tumeurs humaines prdsentent une distribution anormale de I'ADN. Le cardnome in situ du col utdrin montre la m#me distribution de I ' A D N que dam la tumeur invasive. Ce phe'nomkne a aussi pu gtre suivi aux diffdrents stades d'invasion des tumeurs. Le processus atypique de l'hydrolyse, dans la rdaction de Feulgen, au niveau des cellules tumorales, peut gtre mis en relation avec une sensibilit~ diff#entielle, a lygard des acides, de l'euchromatin et de l'hdtgrochromatin.

SUMMARY

A short account ofcytophotometric investigations on malignant and on early cancerous lesions is given. It. was possible to show, that human tumours exhibit an atypical D N A distribution pattern (DNA stem line). The cardnoma in situ of the exocervix shows similar as in malignant tumors a DaVA stem line. This phenomenon can be traced also in the stage of invasion. The atypical course of Feulgen hydrolysis in tumour cells is related to a differential sensivitity towards acids of eu- and heterochromatin.

ZUSAMMENF&SSUNG

Es wird ein kurzer Uberblick der eigenen cytophotometrischen Messungen an malignen Tumoren und Krebsvorstadien gegeben. Es wurde gezeigt, da~ die meisten menschlichen Tumoren ein atypisches DNS-Verteilungsmuster aufweisen (DNS-Stammlinie). Das Carcinoma in situ der Portio zeigt ebenfalls DNS-Stammlinien, die auch bei der Invasion nachzuweisen sin& Atypische Feulgenhydrolysekurven bei TumorzeUen werden auf eine unterschiedliche Sdureempfindlichkeit von Eu und Heterochromatin bezogen.

REFERENCES 1. W. SANDRITTER, D. MOLLER and O. GENSECmS, UV-mlkrospektrophotometrische Messungen des Nukleins~iuregehaltes von Spermien und diploiden Zellen. Aaa Histochem. 10, 139-154 (1960). 2. N. B ATtar, Nuclear size on carcinoma of the cervix: Its relation to DNA content and to prognosis. Cancer17, 1391-1399 (1964) 3. W. SA~RITT~R and D. KI~mHANS, Uber das Trockengewicht, den DNS- und Histonproteingehalt von menschlichen Tumoren. Z. Krebsforsch. ~ , 333-348 (1964). 4. W . SANDRITTER, A. SEmEL, D. KLEINHANS, I. PADDAGS and W . DONTENWILL, Zytophotometrische Messungen des DNS-Gehaltes an menschlichen und tierexperimentellen Bronchialmetaplasien. Z. Ifrebsforsch. (In press). 5. W. SANDRITTER, Cytophotometrische Untersuchungen am Portiocarinom und seinen Vorstufen. Verb. Dtsch. Ges. Path. 48. Tgg. Salzburg, 34-43 (1964). 6. A. S1~.IDEL and W. SANDRITTER, Cytophotometrische Messungen des D N S Gehaltes eines Lungenadenomas und einer malignen Lungenadenomatose. Z" Krebsforsch. 65, 555-559 (1963) 7. W. SArcORrrrEg, I. HILLWm, K. ENOELB.~tT, G. KIm~ER and R. KaSFER, Versuche zur Carcinogenese in vitro. Chromosomenanalysen und cytophotometrische Messungen des DNS-Gehaltes Z. Ifrebsforsch. (In press). 8. W. Smcom'rr~R and N. BOH~, Atypische Hydrolysekurven bei der Feulgenreaktion yon M~iuseascitestumorzellen. aVaturwissenschaften51, 273 (1964). 9. W. SArcoma'rER, K. JOBST, L. RAXOW, and K. BOSSP.LMANN,Zur Kinetik, der Feulgenreaktion bei verlangerter Hydrolsezeit. Cytophotometrishce Messungen im sichtbaren und ultravioletten Licht. Histochemie 4, 420-437 (1965).

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