A STUDY OF MITOSIS IN CERVICAL EPITHELIUM DURING

A Study of Mitosis in Cervical Epithelium during Experimental Inflammation and Carcinogenesis* D. G. SCARPELLI!ANDE. VONHAAM (Department of Pathology, Ohio State University, Columbus, Ohio) spindle mechanism, sections were stained by the van Gieson (19) technic. Serial sections of uterine cervix were examined with an oilimmersion objective at a magnification of 970 diameters. The stage and type of mitosis were determined in 100 dividing epithelial cells in each animal. To insure a more representative sampling of mitoses, numerous regions of the cervical epi thelium were examined. Morphologic criteria characteristic of the various stages of mitosis were followed closely. Cells in which the mitotic stage was not readily ascertainable were not considered in this study. To determine the relative duration of prophase and metaphase, the prophase index was calculated for each animal by dividing the number of metaphases by the number of prophases according to the method of Timonen and Therman (18). These data were subjected tÂ»statistical analysis by the Statistics Laboratory of the Ohio State Uni versity.

Since the early descriptions of abnormal mito sis in malignant cells by von Hansemann (6) and Pianese (17), numerous detailed cytological stud ies of this phenomenon have appeared. The ma jority of these investigations deal with the classi fication of abnormal mitotic types and the pos sible mechanism involved (10). Although abnor mal mitoses were also studied during inflammation by Kemp in 1930 (8) and more recently in carci noma in situ by Parmentier and Dustin (16), and Hamperl et al. (5), no information is available on mitotic abnormalities during the pathogenesis of cancer. The present paper represents a compara tive study of the incidence and types of mitotic abnormalities in epithelial cells of the uterine cer vix in C3H mice during experimental inflamma tion and the induction of cervical carcinoma. MATERIALS

AND METHODS

In total, 50 C8H female mice were selected for the present study. These consisted of four groups of ten animals each, showing the following lesions of the uterine cervix: inflamma tion, dysplasia, carcinoma in situ, and invasive cancer. A fifth group consisting of ten normal C8H mice was used as controls. Inflammation was produced by painting mice intravaginally with a 4 per cent solution of croton oil in acetone. Epithelial dysplasia and cervical carcinoma were produced by painting the animals intravaginally with a 1 per cent solution of 3,4benzpyrene in acetone. The technic of painting is described in detail in a previous communication (4). Smears were prepared previous to each painting procedure and were stained by the method of Papanicolaou (15). As soon as a definite diagnosis of inflammation, dysplasia, or carcinoma was made, the animals were sacrificed. The uterus and vagina were removed, fixed in 10 per cent buffered formalin, and serial sections (5 and 10 /*) were prepared for microscopic examination. In addition to the routine hematoxylin and eosin stain, selected sections were sub jected to the Feulgen reaction (8) for the demonstration of chromosomes. Tissue sections were hydrolyzed in N HC1 at 60Â°C. for 12 minutes, followed by staining in leukobasic fuchsin for lj hours. In order to more clearly demonstrate the * Presented in part at the International Cytology Congress, Chicago, 111.,October 11, 1956. This study was aided by grant C-2704-R from the National Institutes of Health, I'.S. Public Health Service. t National Pathology.

Science Foundation

Postdoctoral

Received for publication May 13, 1957.

Fellow in

RESULTS The mitotic aberrations found in 5000 divid ing cells in normal cervical epithelium, during in flammation, and in the various stages of carcinogenesis are shown in Table 1. A marked differ ence in the number of abnormal mitoses was found between inflammatory and precancerous cervical lesions and cervical cancer. Among the nonmalignant cells showing mitotic abnormality, the pro portion was 4.55 with a standard deviation of 0.47 per cent. Among the malignant cells, the proportion showing mitotic abnormality was 31.15 with a standard deviation of 1.04 per cent. The two proportions were significantly different, with a probability of error less than .001. Normal epithelial cells.â€”Noinstances of abnor mal mitosis such as multipolarity, chromosomal lagging, hollow spindles, asymmetrical divisions, or polar chromosomes were encountered in a study of 1000 dividing cells in the cervical epithelium of normal C3H mice. The number of cells in prophase was roughly equal to those in metaphase. The prophase index varied from .73 to 1.23 with a mean value of .96. Inflammation.â€”Twenty-eight cells with evi dence of abnormal mitosis were found in 1000 dividing epithelial cells in inflammatory lesions of the uterine cervix. Twenty-one of these showed lagging chromosomes with numerous examples of

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SCARPELLIAND VON HAAMâ€”Mitosis during Inflammation bridge formation (Fig. 1). Seven cells showing asymmetrical mitosis were also encountered. The prophase index varied from 0.82 to 1.64 with a mean value of 1.42. Dysplasia.â€”Sixty-three cells showing abnormal mitosis were observed in 1000 dividing cells in cervical dysplasia. Fifty-two examples of lagging chromosomes, seven cells showing hollow metaphase plates (ring forms), and four instances of asymmetric mitosis were found (Fig. 2). The prophase index varied from 1.19 to 2.03, with a mean value of 1.77. Carcinoma in situ.â€”Two hundred and sixtynine cells showing abnormal mitosis were found in 1000 dividing cells in carcinoma in situ of the uterine cervix. One hundred and twelve cells showed lagging chromosomes, 29 showed multipolar mitoses (Figs. 3-5), 23mitotic figures showed hollow metaphase plates (Figs. 6, 7), four of which

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ing the pathogenesis of cervical cancer. The rela tive frequencies of the various mitotic stages and of abnormal mitoses are summarized in Chart 1. An increase in the frequencies of metaphases and a decrease in the frequencies of prophases occurred during the various stages of carcinogenesis. The frequencies of anaphase and telophase did not vary appreciably. A direct relation appears to ex ist between the number of abnormal mitoses and the prophase index, as shown in Chart 2, where the logarithm of the prophase index was plotted against the number of abnormal mitoses. An in crease in both the prophase index and number of abnormal mitoses occurred in malignant lesions, which sharply differentiated them from nonmalignant lesions. Although the prophase indices of the various groups showed a considerable range of values, the prophase index (PI) in normal mice approximated

TABLE 1

INCIDENCEANDTYPESOFNORMALANDABNORMAL MITOSESIN 5,oooDIVIDINGCELLSOFTHE UTERINECERVIX MITOSES

NOBMAL EPITHELIUM

Infiammation

Multipolar Hollow metaphase Asymmetric division Lagging chromosomes Polar chromosomes Normal

O O 0 0 O 1000

O O 7 21 O 972

TOTALS

1000

1000

CEBVICALLESIONS Carcinomain Dysplasia silu29234711258781Invasivecarcinoma52556113749646 O 7 4

52 O 937

1000

1000

1000

were incomplete ring forms (Fig. 8), and 47 in a ratio of 1, while in inflammation, dysplasia, stances of asymmetrical mitosis were found. In carcinoma in situ, and invasive carcinoma the PI addition, 58 cells showed the presence of chromo values were always greater than 1. Since the PI somes distributed about both centrosomes (polar in normal mice tended to approach 1, PI data chromosomes) (Fig. 9). The prophase index varied from all the groups were subjected to statistical from 1.85 to 9.6, with a mean value of 7.3. analysis by testing at various significance levels Invasive carcinoma.â€”Three hundred and fifty- the hypothesis that the prophase index = 1 (i.e., four abnormal mitoses were encountered in 1000 p = Ã¨,where p = the conditional probability that dividing cells in invasive carcinoma of the uterine a cell is in metaphase). Tables of critical PI values cervix. One hundred and thirty-seven cells showed were constructed where the critical PI value was lagging chromosomes, 55 cells showed hollow meta that value above which further increases were of phase plates, eleven of which showed incomplete diagnostic significance at various levels of prob ring formation, 52 showed multipolar mitoses, 47 ability. The results showed that in a sample as showed asymmetric mitosis, and 49 cells showing small as 64 cells, composed of cells either in propolar chromosomes were found (Fig. 10). In sev phase or metaphase, a critical PI value of 2.56 eral instances polar chromosomes appeared to be or greater was diagnostic of malignancy, with a composed of two distinct chromosomal masses probability of error less than .0005. The number (Fig. 11). Occasional cancer cells showed widely of animals with PI values greater than critical PI scattered chromosomes, the so-called "colchicine values at various levels of significance are shown effect" (Fig. 12). The prophase index varied from in Table 2. At a low level of significance (P< .05), 1.79 to 24, with a mean value of 11.2. seven of ten animals with dysplasia had significant A progressive decrease in the relative frequency PI values; however, when the level of significance of the prophase stage of mitosis was evident dur- was increased to a probability of error less than


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.0005, none of these values was significant. On the other hand, eighteen of twenty animals with either carcinoma in situ or invasive carcinoma showed significantly increased PI values with a probability of error less than .0005. DISCUSSION Cytological analysis of mitotic abnormalities in cervical epithelial cells during inflammation

and the pathogenesis of 3,4-benzpyrene-induced cervical carcinoma in C3H mice revealed a sta tistically significant increase in the number of ab normal mitoses as the malignant state was ap proached. Lagging chromosomes constituted the most common abnormality found and were pres ent in inflammation, dysplasia, carcinoma in situ, and invasive carcinoma. Lagging has been attrib uted by various investigators to an increased

PROPHASE

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0PNORMAL

|â€”¿

PMAT INFLAMM.

PMAT DYSPLASIA

PMAT CA. IN SITU

PMAT INVASIVE CA

CHART1.â€”Theaverage frequency of mitotic stages in normal animals and the four types of cervical lesions

60

.NORMAL CO

so

+ INFLAMMATION ODYSPLASIA

o

â€¢¿ CARCINOMA Â¿INVASIVE

IN SITU CANCER

o: O Z

tu 20

O LU

f

I0

a

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â€¢¿ â€¢¿ o +44- O

Ã•8 Ã•9 00

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02

LOG

03

04

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PROPHASE

05

06

07

08

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1.0

I.I

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1.3

1.4

INDEX

CHART2.â€”Scattergram showing a correlation between abnormal mitoses and the prophase index


SCARPELLIAND VON HAAMâ€”Mitosis during Inflammation "stickiness"

of chromosomes, which delays and

modifies chromosomal alignment and separation (1,9). Although numerous examples of asymmetrical division were observed, direct cytological evidence of nondisjunction was obtained only in a few in stances of extremely unequal division. The occa sional presence of micrometaphase figures sug gests that some of the asymmetrical mitoses result in cells which may be viable and capable of fur ther division. Multipolar mitoses constituted the most fre quent multipolar anomaly, although both quadripolar and pentapolar forms were also present. Moorhead and Hsu (14) have shown by cinemato graphic studies of HeLa cells in tissue culture that multipolar spindle formation is the result of in complete fusion of two or more separate spindles. Other workers explain multipolarity on the basis of desynchronization of the various processes that constitute the mitotic cycle. More specifically, they attribute multipolarity to a continued di vision of centrosomes, while the chromosomes di vide only once (18). Polar chromosomes identical with the "metaphase Ã trois groupes" described in human cases of cervical cancer by Parmentier and Dustin (16) were found in experimental cervical carcinoma in mice. Contrary to their findings, this peculiar mi totic anomaly was found with equal frequency both in noninvasive and invasive carcinoma. Von Moellendorff (13) reported similar polar chromo somal aggregations in tissue cultures of fibroblasts following treatment with either polycyclic carcino gens, estrogenic, or androgenic hormones. Although the exact mechanism of polar chromosome for mation is not known, experimental studies by Dustin and Parmentier (2) have shown that it is probably related to chromosome rupture and is not associated with disturbances of the spindle mechanism. The exclusive presence of polar chro mosomes in dividing malignant epithelial cells suggests a possible relationship between this mi totic anomaly and the malignant state. The PI values found in normal and neoplastic cervical tissues of the mouse are of the same mag nitude and range as those observed by Timonen and Therman (18) in their study of human cervi cal cancer cells. In the mouse, PI values greater than 2.17 were found in experimentally induced carcinoma of the uterine cervix. When these data were subjected to rigid statistical analysis, PI values of this magnitude were found to be highly significant. This critical PI value is higher than that of 1.5 reported for human cervical cancer by Timonen and Therman (18) and is no doubt due

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88!Ã®

to differences in the duration of the mitotic cycle of the mouse. The increase in the PI values during the devel opment of cervical carcinoma suggests a correla tion between a shortening of the prophase and/or a prolongation of the metaphase and malignancy. However, owing to the fact that the present data were obtained from fixed tissues, it was impossible to determine exactly where the change occurred in the mitotic cycle. The observations of previous workers (11, 12) that anomalous mitoses require more time for completion than do normal ones may explain the direct relation between the fre quency of abnormal mitoses and increased PI val ues (Chart 2). In view of recent reports (7, 14) that early prophase is not readily recognizable in fixed and stained cells, prophase indices determined from fixed tissues may be in error. Hsu also showed that a discrepancy exists between the prophase TABLE 2

STATISTICAL SIGNIFICANCE OF THE PROPHASEINDEX IN VARIOUS CERVICAL LESIONS Lesions

Normal (controls) Inflammation Dysplasia Carcinoma in situ Invasive carcinoma

No. animals with prophase indices (PI) greater than critical PI values at specified significance levels

P<.OS 000 000 760 10 10

P<.005

10 9

P<.0005

0 9

coefficients (prophases divided by metaphase) ob tained from a study of fixed cells and those ob tained from living cells (7). His results serve to emphasize not only the difficulties encountered when a dynamic process, such as cell division, is studied by static methods utilizing dead cells, but also the need for a re-evaluation of mitotic phe nomena by cinematographic studies of living cells. Previous observations (4) indicated that, al though both qualitative and quantitative cyto logical changes occurred during the malignant transformation of squamous cells, they were grad ual and roughly paralleled the histological altera tions seen in the various stages of carcinogenesis. Our present studies show that disturbances in the metaphase/prophase ratio during the pathogenesis of cancer is not gradual and sharply differen tiates malignant from nonmalignant lesions. The similarity between carcinoma in situ and invasive carcinoma with reference to mitotic abnormalities and prophase indices lends further support to the concept that both lesions are closely related and that circinoma in situ probably represents a ma lignant lesion.


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It should be emphasized, however, that our data are applicable only for experimental carci noma in situ and invasive carcinoma in the mouse cervix. SUMMARY 1. Although a few mitotic abnormalities were found in inflammatory cervical lesions, ever in creasing numbers were observed during the pathogenesis of experimentally induced cervical can cer. 2. Lagging chromosomes constituted the most frequent mitotic abnormality. Polar chromosomes were found only in malignant cervical lesions. 3. Changes in the frequency and/or length of prophase and metaphase were observed which sharply differentiated benign from malignant le sions during the pathogenesis of cancer. 4. The critical prophase indices which were di agnostic of malignancy ranged from 2.15 to 2.56 or above, depending on the number of either prophase or metaphase mitoses counted.

cinoma of the Cervix: A Comparative Cytologie and Histologie Study. Cancer Research, 15:449-55, 1955. 5. HAMPERL,H.; KAUFMANN,C.; and OBER, K. G. Histologische Untersuchungen an der Cervix schwangerer Frauen. Die Erosion und das Carcinoma in situ. Arch. f. GynÃ¤k.,184:181-280, 1954. 6. HANSEMANN, D. VON.Ueber asymmetrische Zelltheilung in Epithelkrebsen und deren biologische Bedeutung. Virchow's Arch. f. path. Anat., 119:299-326, 1890.

1924. 4. HAAM,E. VON,and SCARPELLI,D. G. Experimental Car

7. Hau, T. C. Cytologieal Studies on HeLa, a Strain of Human Cervical Carcinoma. 1. Observations on Mitosis and Chromosomes. Texas Rep. Biol. & Med., 12:833Ht6, 1954. 8. KEMPT,T. Ueber die somatischen Mitosen bei Menschen und warmblutigen Tieren unter normalen und patholo gischen VerhÃ¤ltnissen.Ztschr. f. Zellforsch, u. mikr. Anat., 11:429-44, 1930. 9. KOLLER,P. C. Origin of Malignant Tumor Cells. Nature, 161:244-46, 1943. 10. . Abnormal Mitosis in Tumors. Brit. J. Cancer, 1: 38-47, 1947. 11. LAMBERT,R. A. Comparative Studies upon Cancer Cells. II. The Character of Growth in Vitro with Special Refer ence to Cell Division. J. Exper. Med., 17:499-510, 1913. 12. LEWIS, M. R., and LEWIS, W. H. Malignant Cells of Walker Rat Sarcoma No. 338. Am. J. Cancer, 16:1153-83, 1932. 13. MOELLENDORFF, W., VON.Zur Kenntnis der Mitose. VIII. Zur Analyse des pathologischen Wachstums hervorgerufen durch Chloralhydrat, Geschlechtshormone und can cerogene Kohlenwasserstoffe. Ztschr. f. Zellforsch, u. mikr. Anat., 29:706-49, 1939. 14. MOORHEAD,P. S., and Hau, T. C. Cytologie Studies of HeLa, a Strain of Human Cervical Carcinoma. III. Dura tions and Characteristics of the Mitotic Phases. J. Nat. Cancer Inst., 16:1047-66, 1956. 15. PAPANICOLAOÃœ, G. N. A New Procedure for Staining Vaginal Smears. Science, 96:438-39, 1942. 16. PARMENTIER,R., and DUSTIN, P., JR. Reproduction ex pÃ©rimentaled'une anomalie particuliÃ¨re de la metaphase des cellules malignes (metaphase "Ã trois groupes"). Caryologia, 4:98-109, 1951. 17. PIANESE, G. Beitrag zur Histologie und Aetiologie des Carcinoms. Ziegler's BeitrÃ¤gez. path. Anat., 1:193, 1896. 18. TiMONEN, S., and THERMAN,E. The Changes in the Mitotic Mechanism of Human Cancer Cells. Cancer Re search, 10:431-39, 1950. 19. VANGIBSON,I. Laboratory Notes of Technical Methods for the Nervous System. New York M. J., 50:57-60,1889.

All microphotographs were taken at a magnification of 1300 diameters. Unless otherwise stated, all were stained with hematoxylin-eosin. FIG. 1.â€”Bipolarmitosis showing lagging chromosomes. FIG. 2.â€”Bipolar anaphase showing asymmetrical division of chromosomes. FIG. 3.â€”Tripolar metaphase. FIG. 4.â€”Tripolar anaphase. FIG. 5.â€”Tetrapolar metaphase showing numerous spindles (van Gieson stain). FIG. 6.â€”Hollowmetaphase plate.

FIG. 7.â€”Micronucleusshowing a hollow metaphase mitosis. FIG. 8.â€”Incomplete hollow metaphase plate. FIG. 9.â€”Metaphase showing polar chromosomes. FIG. 10.â€”Metaphase showing polar chromosomes. Two chromosome masses are present at the upper pole (Feulgen re action). FIG. 11.â€”Metaphase showing polar migration of a chromo somal mass. FIG. 12.â€”Malignant squamous cell in division showing the so-called "colchicine effect." Note the widely scattered and contracted chromosomes.

ACKNOWLEDGMENTS The authors are indebted to Dr. D. Ransom Whitney, Statistics Laboratorjr, Ohio State University, for statistical analysis of the data. REFERENCES 1. DARLINGTON,C. D. Chromosome Chemistry and Gene Action. Nature, 149:66-69, 1942. 2. DUSTIN,P., and PABMENTIER, R. DonnÃ©es experimentales sur la nature des mitoses anormales observÃ©es dans cer tains epitheliomas du col utÃ©rin.Gynecol. et Obst., 52: 258-65, 1953. 3. FEULGEN, R., and ROSSENBECK,H. Mikroskopischchemischer Nachweis einer NucleinsÃ¤urevon Typus der ThymonucleinsÃ¤ure und die darauf beruhende elektive FÃ¤rbungvon Zellkernen in mikroskopischen PrÃ¤paraten. Hoppe-Seyler's Ztschr. f. physiol. Chem., 135:203-48,



A Study of Mitosis in Cervical Epithelium during Experimental Inflammation and Carcinogenesis D. G. Scarpelli and E. von Haam Cancer Res 1957;17:880-884.

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A STUDY OF MITOSIS IN CERVICAL EPITHELIUM DURING

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