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Differential chromosomal effects of carcinogenic and noncarcinogenic substances
Differential
chromosomal
and noncarcinogenic An experimental
HAROLD
A.
GEORGIANA Chicago,
study
substances
on the mouse
KAMINETZKY, M.
effects of carcinogenic
JAGIELLO,
M.D. M.D.
Illinois
In the mouse, lesions resembling carcinoma in situ are induced by repeated topical application of methytchotanthrene and podophyttin. Methylcholanthrene is a known carcinogen, podophyttin is not. The present study was undertaken to compare the chromosomat effects of these substances. A single application of methytcholanthrene induced no discernible differences when compared with controls. A single application for podophyttin produced numerous mitotic figures. All but two were metaphases and all nuclei contained the species chromosome number. Among the animals receiving repeated methytchotanthrene applications, most chromosomes appeared morphologically identical to the controls; however, a number of metaphase figures had grossly distorted chromosomes and appeared dead or dying or entirely beyond the usual mitotic controls. Among the animals receiving repeated podophyltin applications a large number of nuclei contained twice the species chromosome number (80). Chromosome coalescence was found only in this group and probably is the phenomenon responsible for the malignant appearance of podophyllin-treated cells.
L E s I o N s resembling spontaneous cervical dysplasia and carcinoma in situ are produced in mice by vaginal application of a suspension of podophyllin in mineral oi1.4 Exfoliated cells recovered in corresponding vaginal smears often are indistinguishable from those of epidermoid carcinoma. On the other hand, multiple applications of this substance over many months failed to induce invasive carcinoma.3* 5 By comparison, true chemical carcinogens such as methylcholanthrene produced similar-appearing intraepithelial lesions and exfoliated cells and, ultimately, invasive carcinoma. The purpose of the present study was to determine what,
if any, differences might be found mosome analysis of cells affected substance. Materials The mice strain.
and methods in this study
were
by chroby each
of the C&H
Experimental Plan. 1. The control group consisted of 10 untreated animals used to gain familiarity with the species chromosome number and general morphology. 2. A single application of a 1 per cent suspension of methylcholanthrene in mineral oil was applied to the cervix and vagina of 25 mice. One mouse was killed hourly. 3. A single application of a 10 per cent suspension of podophyllin in minera oil was applied to 25 mice as in Experiment 2. Experiments 2 and 3 were designed to determine the acute effects of a single application of each substance. Previous work in-
From the Departments of Obstetrics and Gynecology and Medicine, University of Illinois at The Medical Center. Presented at the Thirty-fourth Annual Meeting of the Central Association of Obstetricians and Gynecologists, Biloxi, Mississippi, Oct. 20-22, 1966.
349
350
Kaminetzky
and
June 1, 1967 Am. J. Obst. & Gyn~c.
Jagiello
Fig. 1. A typical metaphase figure containing chromosomes found in the tissue preparation one of the control animals.
Fig. 2. A metaphase figure from a mouse ing 19 methylcholanthrene applications. are 40 chromosomes of normal appearance.
40 from
followThere
Fig. 3. A disorganized chromosome found in a tissue spread following anthrene applications.
Fig. 4. A bizarre mouse following tions.
chromosome group 19 meth$&&rr~rm
conglomerate 19 methylchol-
from
another ap@iea-
Volume Number
9tl 3
Chromosomal
dicated podophyllin affects chromosomes within several hours after a single application.’ 4. One per cent methylcholanthrene in mineral oil was applied twice weekly to the cervix and vagina of 30 mice for a total of 19 to 20 times prior to being killed. 5. Ten per cent podophyllin in mineral oil was applied twice weekly to the cervix and vagina of 30 mice for a total varying from 19 to 22 times prior to being killed. Experiments 4 and 5 were designed to compare the chromosome effects of each substance at a time when previous experiments indicated there would likely be a marked squamous epithelial effect. To facilitate chromosome analysis in the control and methylcholanthrene-treated mice, 0.1 C.C. of an 0.5 per cent aqueous colchicine solution was injected intraperitoneally 55 minutes prior to killing to arrest mitosis. This was not necessary with the podophyllintreated mice because this substance itself possesses the property of arresting mitosis. All mice were killed by acute displacement of the cervical spine, to avoid possible chromosome alterations induced by anesthesia. Then, the vagina and cervix were dissected free and opened sagittally; 10 to 15 small fragments were clipped at random from the epithelial surfaces and processed by the following modification of the method of Ohno. The fragments were immersed in distilled water for 15 minutes and transferred to a solution of 50 per cent acetic acid for 15 minutes. Each fragment was then placed on a clean microscope slide in several drops of the acetic acid solution and pounded repeatedly with the blunt end of a metal rod to separate the epithelial cells from each other. A cover slip was placed over the suspension and a tissue squash made applying pressure with both thumbs, The covered squash was frozen in methyl alcohol chilled by floating carbon dioxide ice. The cover slip was removed and the slide allowed to dry in air. The adherent residuum was treated with Giemsa stain. All slides were screened with a Zeiss photomicroscope through a 40x eyepiece, an
effects
of methylcholanthrene
and
podophyllin
351
Fig. 5. Distorted chromosomes from a tissue squash following 20 methylcholanthrene applications.
Fig. 6. Abnormal cells found in a preparation from a mouse after 20 methylcholanthrene applications.
352
Kaminetzky
and Jagiello
June 1, 1967 Am. J. Obst. & Gym.
8~ objective, and the 1.2 Optovar lens. To avoid chromosome interlopers, the nuclei analyzed were those that appeared intact and remote from broken nuclei. Nuclei were photographed through the oil-immersion objective on 35 millimeter Kodak high-contrast film. Following development, the negatives were enlarged 9 times on glossy photographic printing paper. Chromosome counts were made by dividing the print of each mitotic figure into four parts, counting the number of chromosomes in each part, and adding. Results
Fig. 7. Eighty chromosomes found following 19 podophyllin applications.
in
Fig. 8. Chromosome coalescence found cleus following 19 podophyllin applications.
a nucleus
in
a nu-
1. Few mitotic figures were found in the tissue from the control animals. The species chromosome number is 40. Fig. 1 is a photomicrograph of a typical metaphase figure. 2. A single application of methylcholanthrene produced no discernible differences when compared with the controls. Few mitotic figures were found; the chromosomes appeared unchanged morphologically and each nucleus contained the species chromosome number. 3. A single application of podophyllin produced numerous countable mitotic figures by comparison with controls. The effect was well established at 5 hours when 10 metaphases were found. The chromosomes from a total of 47 cells were countable among the 25 mice in this group; all but 2 were metaphase figures. At 19 and 20 hours, respectively, single telophase and anaphase figures were found possibly indicating escape from the effect of podophyllin. All cells contained 40 chromosomes which appeared morphologically identical to those of the controls. 4. In the group of animals receiving 19 to 20 applications of methylcholanthrene, a total of 75 cells were found suitable for analysis. Sixty-one cells contained 40 chromosomes (Fig. 2)) 11 contained 41 chromosomes, and 3 contained fewer than 38 chromosomes. Most chromosomes appeared morphologically identical to those of the controls ; however, a number of cells had grossly distorted chromosomes that appeared to be dead, dying or entirely beyond the
Volume Number
98 3
Chromosomal
usual orderly mitotic controls (Figs. 3, 4, and 5). Alone in this group were seen numerous intact cells containing large dark nuclei with bluish staining cytoplasm suggesting malignant cells (Fig. 6). 5. Among preparations of the animals receiving 19 to 22 podophyllin applications the most striking feature was the large number of cells containing nuclei with exactly twice the species chromosome number (80) (Fig. 7) . Among 185 cells counted, 78 contained 40 chromosomes, 72 contained 80 chromosomes, 14 contained 41 chromosomes, 11 contained 39, 2 contained 42, 1 contained 62, and 7 contained fewer than 38 chromosomes. Coalescence of chromosomes was found only in preparations from this group of animals (Fig. 8) . Following coalescence, the nuclei appear malignant. Comment Clinical experience indicates that in some patients, cervical dysplasia and carcinoma in situ disappear without specific treatment, in others there is evidence of progression from dysplasia through carcinoma in situ to invasive cancer. Speculatively, epithelial atypia can arise from two classes of stimuli, one leading to invasive cancer, the other producing similar-appearing intraepithelial lesions incapable of invasion. Presently, there are no morphologic or biochemical criteria for predicting the outcome of any atypical lesion. Contrariwise, the fate of an atypical lesion may not depend necessarily on the nature of the original stimulus but rather on some unidentified factor or factors within
effects
of
methylcholanthrene
and
podophyllin
353
the host that control stromal penetration by abnormal cells. The study reported here addressed itself to the possibility there are really two different classes of stimuli and this to the best of our knowledge is the first such study. The results of these experiments suggest that cells of similar appearance can be produced by different chromosome effects. Interestingly, doubling of the chromosome number induced by podophyllin is reported frequently in spontaneous malignant tumors and in carcinoma in situ, yet this effect was not seen in cells affected by methylcholanthrene. Both substances induced considerable variation in the nuclear chromosome number; spontaneously arising malignant neoplasms also exhibit this feature.l, 6p g, lo The fact that podophyllin application does not produce invasive carcinoma suggests that many of the cellular features of malignancy can be produced by a noncarcinogen. There may be naturally occurring substances that can some metabolite of do likewise ; possibly, Trichomonas vaginalis acts in this manner. Presently, the nature of the cellular changes leading to invasive cancer are unknown. Grossly visible modifications of chromosomes may be effects of, rather than the cause of malignancy. It seems reasonable that invisible and at present chemically undetectable modifications of nuclear or cytoplasmic DNA or RNA may so alter protein synthesis as to give rise to malignancy which in turn may alter chromosome number and morphology secondarily.2
REFERENCES
1. 2. 3. 4. 5. 6.
Boddington, M. M., Spriggs, A. I., and Wolfendale, M. R.: Brit. M. J. 1: 154, 1965. Hauschka, T. S.: Exper. Cell Res. Suppl. 9: 86, 1963. Kaminetzky, H. A., and McGrew, E. A.: Arch. Path. 73: 481, 1962. Kaminetzky, H. A., McGrew, E. A., and Phillips, R. L.: Obst. & Gynec. 14: 1, 1959. Kaminetzky, H. A., and Swerdlow, M.: AM. T. OBST. & GYNEC. 93: 486. 1965. Kirkland, J. A.: Australia New Zealand J. Obst. & Gynaec. 6: 35, 1966.
7. 8. 9. 10.
McGrew, E. A., and Kaminetzky, H. A.: Am. J. Clin. Path. 35: 538, 1961. Ohno, S.: Personal communication. Wakonig-Vaartaja, R., and Hughes, D. T.: Lancet 2: 756, 1965. Wakonig-Vaartaja, R., and Kirkland, J. A.: Cancer 18: 1101, 1965. 840 South Wood P.O. Box 6998 Chicago, Illinois
Street 60680
354
Kaminetzky
and
Jagiello
Discussion Rochester, Minnesota. With a clearly defined experiment, Drs. Kaminetzky and Jagiello have added another report to a series of investigations relative to carcinoma of the cervix. A new technique, chromosome analysis, has been used to evaluate the effects of two compounds known to influence cervical epithelium in the mouse. Apparently, a difference in response has been demonstrated. Of 75 cells observed after stimulation with methylcholanthrene, 81.3 per cent remained diploid. Subsequent to exposure to podophyllin, however, only 42.1 per cent of 185 cells were diploid, while 38.9 per cent were tetraploid. Curiously, though, aneuploidy approximated 19 per cent in both groups, a situaDR.
ELIZABETH
MUSSEY,
tion which raises doubt as to the significance of the apparent difference. Despite meticulous use of a standard technique of tissue preparation, such as that described, unsatisfactory preparations are said to be frequent, with chromosomes too poorly defined to count. One wonders if the chromosomal aberrations exhibited in Figs. 3, 4, and 5 are manifestations of technical difficulties. It is interesting that the cervices treated with methylcholanthrene yielded only 75 cells suitable for analysis, whereas 185 cells were obtained from the same number of podophyllin-treated animals. Could primary drug toxicity, rather than oncogenesis, be responsible for this inequality? The importance of the findings as they relate to the clinical problem of human cancer of the cervix is obscure. Aneuploidy exceeding the normal incidence of 12 to 15 per cent has been found consistently in reported studies of lesions of the human cervix. The variations in numbers of chromosomes have not followed any pattern, although a diploid mode appears to be preserved in chromosome counts from dysplasia. Polyploidy is more evident in preinvasive and invasive carcinoma than in benign atypia. Aneuploidy is not pathognomonic of malignancy, however, for it is found in healthy states as well as in nonmalignant disease. Further, it is not known whether all aneuploid cells are capable of replication or whether tetraploidy, especially, can result from endomitosis, which is not true reproduction. The latter process might cause the polyploidy after podophyllin and might account for the failure of the lesions to progress to cancer. Because of lack of variation in chromosome
June 1, 1967 Am. J. Obst. & Gyn~c.
morphology, karyotyping is difficult to accomplish in cells from the mouse. However, the procedure might yield data which would differentiate the effects of one compound from those of the other. Identification of a so-called marker chromosome, the presence of which serves to establish the existence of pseudodiploidy, would indicate a departure from normal chromosomal pattern in spite of normal numbers. One would like conventional histologic identification of the areas from which the chromosome counts were made in order to assess possible differences in tissue patterns in the cervices of the two groups of mice, but I suspect brevity dictated omission of these data. I am sure that Dr. Kaminetzky agrees that more questions have been raised than answered, and this is appropriate for a pioneer experiment that is a provocative approach to a vexing clinical problem. We can look forward to additional stimulating reports from this group of investigators. DR. GLORIA SARTO, Madison, Wisconsin. I wonder if the tetraploidy, which occurred in 76 of the total number of cells, might have been due to some interference with spindle formation by podophyllin alone. This might cause duplication of chromosomes without separation and division of the cells, resulting in tetraploidy. Insofar as coalescence of chromosomes is concerned, from the preparations shown, it was difficult to tell if this may have been the result of chromosome breaks with interchromosomal reunion, resulting in unusually shaped chromosomes. This phenomenon is interesting and it ought to be investigated further. DR. KAMINETZKY (Closing). Dr. Mussey asked if the specific chromosome abnormalities found in methylcholanthrene-treated mice might not be artifacts caused by faulty technique. Certainly, this is a possibility. When handling tissues from a large number of antimals, technical procedures must of necessity be done on a considerable number of days. However, it seems unlikely that a technical error would involve only one experimental group. Certainly, we tried to keep our technique identical for each group of animals. The reason that tissue treated with podophyllin contained the greatest number of countable nuclei relates to the fact that this substance itself arrests mitosis. We used a relatively small amount of colchicine in the control and methyl-
Volume Number
98 3
Chromosomal
cholanthrene-treated animals to arrest mitosis and anticipated there would be fewer nuclei available for chromosome analysis. This is a limitation of the present experimental method. It is possible that podophyllin induces tetraploidy by the process of endoreduplication and that cells exhibiting this feature are not viable. I would point out that tetraploidy of similar appearance is also found in preparations from spontaneous human in situ and invasive carcinoma. Considering the problem of obtaining tissue for histologic study immediately adjacent to tissue used for chromosomes analysis, I submit that the amount of tissue involved in the mouse is too small to make such studies practical. At the pres-
effects
of
methylcholanthrene
and
podophyllin
355
ent time we are carrying out studies in the rhesus monkey as well as in the mouse. However, even in the monkey, the amount of tissue available for biopsy is small. Possibly, at the end of given experiments the entire cervix can be removed and tissue squashes and histologic specimens can be prepared from contiguous tissue fragments. That chromosome coalescence in the podophyllin-treated animals might be due to chromosome breaks with subsequent reconstitution is a possibility; however, we found no evidence of breaks in any chromosome preparation. There appears to be no valid reason why we should see only the chromosome recombination of but never the break that preceded it.
chromosomal
and noncarcinogenic An experimental
HAROLD
A.
GEORGIANA Chicago,
study
substances
on the mouse
KAMINETZKY, M.
effects of carcinogenic
JAGIELLO,
M.D. M.D.
Illinois
In the mouse, lesions resembling carcinoma in situ are induced by repeated topical application of methytchotanthrene and podophyttin. Methylcholanthrene is a known carcinogen, podophyttin is not. The present study was undertaken to compare the chromosomat effects of these substances. A single application of methytcholanthrene induced no discernible differences when compared with controls. A single application for podophyttin produced numerous mitotic figures. All but two were metaphases and all nuclei contained the species chromosome number. Among the animals receiving repeated methytchotanthrene applications, most chromosomes appeared morphologically identical to the controls; however, a number of metaphase figures had grossly distorted chromosomes and appeared dead or dying or entirely beyond the usual mitotic controls. Among the animals receiving repeated podophyltin applications a large number of nuclei contained twice the species chromosome number (80). Chromosome coalescence was found only in this group and probably is the phenomenon responsible for the malignant appearance of podophyllin-treated cells.
L E s I o N s resembling spontaneous cervical dysplasia and carcinoma in situ are produced in mice by vaginal application of a suspension of podophyllin in mineral oi1.4 Exfoliated cells recovered in corresponding vaginal smears often are indistinguishable from those of epidermoid carcinoma. On the other hand, multiple applications of this substance over many months failed to induce invasive carcinoma.3* 5 By comparison, true chemical carcinogens such as methylcholanthrene produced similar-appearing intraepithelial lesions and exfoliated cells and, ultimately, invasive carcinoma. The purpose of the present study was to determine what,
if any, differences might be found mosome analysis of cells affected substance. Materials The mice strain.
and methods in this study
were
by chroby each
of the C&H
Experimental Plan. 1. The control group consisted of 10 untreated animals used to gain familiarity with the species chromosome number and general morphology. 2. A single application of a 1 per cent suspension of methylcholanthrene in mineral oil was applied to the cervix and vagina of 25 mice. One mouse was killed hourly. 3. A single application of a 10 per cent suspension of podophyllin in minera oil was applied to 25 mice as in Experiment 2. Experiments 2 and 3 were designed to determine the acute effects of a single application of each substance. Previous work in-
From the Departments of Obstetrics and Gynecology and Medicine, University of Illinois at The Medical Center. Presented at the Thirty-fourth Annual Meeting of the Central Association of Obstetricians and Gynecologists, Biloxi, Mississippi, Oct. 20-22, 1966.
349
350
Kaminetzky
and
June 1, 1967 Am. J. Obst. & Gyn~c.
Jagiello
Fig. 1. A typical metaphase figure containing chromosomes found in the tissue preparation one of the control animals.
Fig. 2. A metaphase figure from a mouse ing 19 methylcholanthrene applications. are 40 chromosomes of normal appearance.
40 from
followThere
Fig. 3. A disorganized chromosome found in a tissue spread following anthrene applications.
Fig. 4. A bizarre mouse following tions.
chromosome group 19 meth$&&rr~rm
conglomerate 19 methylchol-
from
another ap@iea-
Volume Number
9tl 3
Chromosomal
dicated podophyllin affects chromosomes within several hours after a single application.’ 4. One per cent methylcholanthrene in mineral oil was applied twice weekly to the cervix and vagina of 30 mice for a total of 19 to 20 times prior to being killed. 5. Ten per cent podophyllin in mineral oil was applied twice weekly to the cervix and vagina of 30 mice for a total varying from 19 to 22 times prior to being killed. Experiments 4 and 5 were designed to compare the chromosome effects of each substance at a time when previous experiments indicated there would likely be a marked squamous epithelial effect. To facilitate chromosome analysis in the control and methylcholanthrene-treated mice, 0.1 C.C. of an 0.5 per cent aqueous colchicine solution was injected intraperitoneally 55 minutes prior to killing to arrest mitosis. This was not necessary with the podophyllintreated mice because this substance itself possesses the property of arresting mitosis. All mice were killed by acute displacement of the cervical spine, to avoid possible chromosome alterations induced by anesthesia. Then, the vagina and cervix were dissected free and opened sagittally; 10 to 15 small fragments were clipped at random from the epithelial surfaces and processed by the following modification of the method of Ohno. The fragments were immersed in distilled water for 15 minutes and transferred to a solution of 50 per cent acetic acid for 15 minutes. Each fragment was then placed on a clean microscope slide in several drops of the acetic acid solution and pounded repeatedly with the blunt end of a metal rod to separate the epithelial cells from each other. A cover slip was placed over the suspension and a tissue squash made applying pressure with both thumbs, The covered squash was frozen in methyl alcohol chilled by floating carbon dioxide ice. The cover slip was removed and the slide allowed to dry in air. The adherent residuum was treated with Giemsa stain. All slides were screened with a Zeiss photomicroscope through a 40x eyepiece, an
effects
of methylcholanthrene
and
podophyllin
351
Fig. 5. Distorted chromosomes from a tissue squash following 20 methylcholanthrene applications.
Fig. 6. Abnormal cells found in a preparation from a mouse after 20 methylcholanthrene applications.
352
Kaminetzky
and Jagiello
June 1, 1967 Am. J. Obst. & Gym.
8~ objective, and the 1.2 Optovar lens. To avoid chromosome interlopers, the nuclei analyzed were those that appeared intact and remote from broken nuclei. Nuclei were photographed through the oil-immersion objective on 35 millimeter Kodak high-contrast film. Following development, the negatives were enlarged 9 times on glossy photographic printing paper. Chromosome counts were made by dividing the print of each mitotic figure into four parts, counting the number of chromosomes in each part, and adding. Results
Fig. 7. Eighty chromosomes found following 19 podophyllin applications.
in
Fig. 8. Chromosome coalescence found cleus following 19 podophyllin applications.
a nucleus
in
a nu-
1. Few mitotic figures were found in the tissue from the control animals. The species chromosome number is 40. Fig. 1 is a photomicrograph of a typical metaphase figure. 2. A single application of methylcholanthrene produced no discernible differences when compared with the controls. Few mitotic figures were found; the chromosomes appeared unchanged morphologically and each nucleus contained the species chromosome number. 3. A single application of podophyllin produced numerous countable mitotic figures by comparison with controls. The effect was well established at 5 hours when 10 metaphases were found. The chromosomes from a total of 47 cells were countable among the 25 mice in this group; all but 2 were metaphase figures. At 19 and 20 hours, respectively, single telophase and anaphase figures were found possibly indicating escape from the effect of podophyllin. All cells contained 40 chromosomes which appeared morphologically identical to those of the controls. 4. In the group of animals receiving 19 to 20 applications of methylcholanthrene, a total of 75 cells were found suitable for analysis. Sixty-one cells contained 40 chromosomes (Fig. 2)) 11 contained 41 chromosomes, and 3 contained fewer than 38 chromosomes. Most chromosomes appeared morphologically identical to those of the controls ; however, a number of cells had grossly distorted chromosomes that appeared to be dead, dying or entirely beyond the
Volume Number
98 3
Chromosomal
usual orderly mitotic controls (Figs. 3, 4, and 5). Alone in this group were seen numerous intact cells containing large dark nuclei with bluish staining cytoplasm suggesting malignant cells (Fig. 6). 5. Among preparations of the animals receiving 19 to 22 podophyllin applications the most striking feature was the large number of cells containing nuclei with exactly twice the species chromosome number (80) (Fig. 7) . Among 185 cells counted, 78 contained 40 chromosomes, 72 contained 80 chromosomes, 14 contained 41 chromosomes, 11 contained 39, 2 contained 42, 1 contained 62, and 7 contained fewer than 38 chromosomes. Coalescence of chromosomes was found only in preparations from this group of animals (Fig. 8) . Following coalescence, the nuclei appear malignant. Comment Clinical experience indicates that in some patients, cervical dysplasia and carcinoma in situ disappear without specific treatment, in others there is evidence of progression from dysplasia through carcinoma in situ to invasive cancer. Speculatively, epithelial atypia can arise from two classes of stimuli, one leading to invasive cancer, the other producing similar-appearing intraepithelial lesions incapable of invasion. Presently, there are no morphologic or biochemical criteria for predicting the outcome of any atypical lesion. Contrariwise, the fate of an atypical lesion may not depend necessarily on the nature of the original stimulus but rather on some unidentified factor or factors within
effects
of
methylcholanthrene
and
podophyllin
353
the host that control stromal penetration by abnormal cells. The study reported here addressed itself to the possibility there are really two different classes of stimuli and this to the best of our knowledge is the first such study. The results of these experiments suggest that cells of similar appearance can be produced by different chromosome effects. Interestingly, doubling of the chromosome number induced by podophyllin is reported frequently in spontaneous malignant tumors and in carcinoma in situ, yet this effect was not seen in cells affected by methylcholanthrene. Both substances induced considerable variation in the nuclear chromosome number; spontaneously arising malignant neoplasms also exhibit this feature.l, 6p g, lo The fact that podophyllin application does not produce invasive carcinoma suggests that many of the cellular features of malignancy can be produced by a noncarcinogen. There may be naturally occurring substances that can some metabolite of do likewise ; possibly, Trichomonas vaginalis acts in this manner. Presently, the nature of the cellular changes leading to invasive cancer are unknown. Grossly visible modifications of chromosomes may be effects of, rather than the cause of malignancy. It seems reasonable that invisible and at present chemically undetectable modifications of nuclear or cytoplasmic DNA or RNA may so alter protein synthesis as to give rise to malignancy which in turn may alter chromosome number and morphology secondarily.2
REFERENCES
1. 2. 3. 4. 5. 6.
Boddington, M. M., Spriggs, A. I., and Wolfendale, M. R.: Brit. M. J. 1: 154, 1965. Hauschka, T. S.: Exper. Cell Res. Suppl. 9: 86, 1963. Kaminetzky, H. A., and McGrew, E. A.: Arch. Path. 73: 481, 1962. Kaminetzky, H. A., McGrew, E. A., and Phillips, R. L.: Obst. & Gynec. 14: 1, 1959. Kaminetzky, H. A., and Swerdlow, M.: AM. T. OBST. & GYNEC. 93: 486. 1965. Kirkland, J. A.: Australia New Zealand J. Obst. & Gynaec. 6: 35, 1966.
7. 8. 9. 10.
McGrew, E. A., and Kaminetzky, H. A.: Am. J. Clin. Path. 35: 538, 1961. Ohno, S.: Personal communication. Wakonig-Vaartaja, R., and Hughes, D. T.: Lancet 2: 756, 1965. Wakonig-Vaartaja, R., and Kirkland, J. A.: Cancer 18: 1101, 1965. 840 South Wood P.O. Box 6998 Chicago, Illinois
Street 60680
354
Kaminetzky
and
Jagiello
Discussion Rochester, Minnesota. With a clearly defined experiment, Drs. Kaminetzky and Jagiello have added another report to a series of investigations relative to carcinoma of the cervix. A new technique, chromosome analysis, has been used to evaluate the effects of two compounds known to influence cervical epithelium in the mouse. Apparently, a difference in response has been demonstrated. Of 75 cells observed after stimulation with methylcholanthrene, 81.3 per cent remained diploid. Subsequent to exposure to podophyllin, however, only 42.1 per cent of 185 cells were diploid, while 38.9 per cent were tetraploid. Curiously, though, aneuploidy approximated 19 per cent in both groups, a situaDR.
ELIZABETH
MUSSEY,
tion which raises doubt as to the significance of the apparent difference. Despite meticulous use of a standard technique of tissue preparation, such as that described, unsatisfactory preparations are said to be frequent, with chromosomes too poorly defined to count. One wonders if the chromosomal aberrations exhibited in Figs. 3, 4, and 5 are manifestations of technical difficulties. It is interesting that the cervices treated with methylcholanthrene yielded only 75 cells suitable for analysis, whereas 185 cells were obtained from the same number of podophyllin-treated animals. Could primary drug toxicity, rather than oncogenesis, be responsible for this inequality? The importance of the findings as they relate to the clinical problem of human cancer of the cervix is obscure. Aneuploidy exceeding the normal incidence of 12 to 15 per cent has been found consistently in reported studies of lesions of the human cervix. The variations in numbers of chromosomes have not followed any pattern, although a diploid mode appears to be preserved in chromosome counts from dysplasia. Polyploidy is more evident in preinvasive and invasive carcinoma than in benign atypia. Aneuploidy is not pathognomonic of malignancy, however, for it is found in healthy states as well as in nonmalignant disease. Further, it is not known whether all aneuploid cells are capable of replication or whether tetraploidy, especially, can result from endomitosis, which is not true reproduction. The latter process might cause the polyploidy after podophyllin and might account for the failure of the lesions to progress to cancer. Because of lack of variation in chromosome
June 1, 1967 Am. J. Obst. & Gyn~c.
morphology, karyotyping is difficult to accomplish in cells from the mouse. However, the procedure might yield data which would differentiate the effects of one compound from those of the other. Identification of a so-called marker chromosome, the presence of which serves to establish the existence of pseudodiploidy, would indicate a departure from normal chromosomal pattern in spite of normal numbers. One would like conventional histologic identification of the areas from which the chromosome counts were made in order to assess possible differences in tissue patterns in the cervices of the two groups of mice, but I suspect brevity dictated omission of these data. I am sure that Dr. Kaminetzky agrees that more questions have been raised than answered, and this is appropriate for a pioneer experiment that is a provocative approach to a vexing clinical problem. We can look forward to additional stimulating reports from this group of investigators. DR. GLORIA SARTO, Madison, Wisconsin. I wonder if the tetraploidy, which occurred in 76 of the total number of cells, might have been due to some interference with spindle formation by podophyllin alone. This might cause duplication of chromosomes without separation and division of the cells, resulting in tetraploidy. Insofar as coalescence of chromosomes is concerned, from the preparations shown, it was difficult to tell if this may have been the result of chromosome breaks with interchromosomal reunion, resulting in unusually shaped chromosomes. This phenomenon is interesting and it ought to be investigated further. DR. KAMINETZKY (Closing). Dr. Mussey asked if the specific chromosome abnormalities found in methylcholanthrene-treated mice might not be artifacts caused by faulty technique. Certainly, this is a possibility. When handling tissues from a large number of antimals, technical procedures must of necessity be done on a considerable number of days. However, it seems unlikely that a technical error would involve only one experimental group. Certainly, we tried to keep our technique identical for each group of animals. The reason that tissue treated with podophyllin contained the greatest number of countable nuclei relates to the fact that this substance itself arrests mitosis. We used a relatively small amount of colchicine in the control and methyl-
Volume Number
98 3
Chromosomal
cholanthrene-treated animals to arrest mitosis and anticipated there would be fewer nuclei available for chromosome analysis. This is a limitation of the present experimental method. It is possible that podophyllin induces tetraploidy by the process of endoreduplication and that cells exhibiting this feature are not viable. I would point out that tetraploidy of similar appearance is also found in preparations from spontaneous human in situ and invasive carcinoma. Considering the problem of obtaining tissue for histologic study immediately adjacent to tissue used for chromosomes analysis, I submit that the amount of tissue involved in the mouse is too small to make such studies practical. At the pres-
effects
of
methylcholanthrene
and
podophyllin
355
ent time we are carrying out studies in the rhesus monkey as well as in the mouse. However, even in the monkey, the amount of tissue available for biopsy is small. Possibly, at the end of given experiments the entire cervix can be removed and tissue squashes and histologic specimens can be prepared from contiguous tissue fragments. That chromosome coalescence in the podophyllin-treated animals might be due to chromosome breaks with subsequent reconstitution is a possibility; however, we found no evidence of breaks in any chromosome preparation. There appears to be no valid reason why we should see only the chromosome recombination of but never the break that preceded it.