Albert Levan

ROLE OF CHROMOSOMES IN CANCEROGENESIS, AS STUDIED IN SERIAL TISSUE CULTURE OF MAMMALIAN CELLS

Basing our views on observations of chromosomal behavior in cancers and following the classical views of Boveri, Winge, and others, we tend to regard the development of malignancy as a process of selection on the cellular level that favors cells with the acquired capacity of escaping the control of the host organism. The regular appearance of new karyotypes in cancer stemlines supports this view. In our opinion the significant feature in some kinds of cancerogenesis is the induction of genotypic variability. Any random genotype variation within a tissue may in time produce cells capable of neoplastic growth. Many facts indicate that the genotypic changes leading to this end are highly complex. Therefore, considerable experimentation with various genotypes will be necessary, and the transition from normal cells over precancerous to malignant cells will be floating. Natural selection within the cell population should automatically lead to cells with ever-greater independence of the host control. Because gross chromosomal disturbances and somatic segregation for chromatid and subchromatid changes will take place a t mitosis, i t is evident that any chromosomal instability will have an especially strong effect in tissues with high mitotic activity. According to this view any mutagenic agent could be carcinogenic, provided the mutagenic action is extended over many mitotic cycles. Even one single exposure to X radiation would thus have a chance to bring about this effect if a specific type of mutation is induced in either the nucleus or the cytoplasm, that is, a mutation giving a permanent increase in mutability in all cells carrying this gene. This view is compatible with the chemical induction of cancer and perhaps will turn out to be so with the viral induction and transfer of the disease. The change in environment connected with the explantation of mammalian cells from their normal position in the body into culture i l z vitro is indeed a drastic one. The fact that a high percentage of cells may survive such a change and may even start reproduction under the new conditions is good evidence of the considerable adaptability of mammalian cells. In many Cornell University Medical College, New I.ork, N . Y .

CHROMOSOMES IN CANCER TISSUE

The cancer literature contains a great number of papers providing information about chromosomes in cancer tissue. Very few of these investigations, however, have chromosomes as their main issue, or are written by cytologists. In the majority of papers, the information about chromosomes is given more or less in passing, as side-products of pathological, chemotherapeutic, or other investigations. Data on cancer chromosomes have been gradually accumulating, however, and it was realized early that chromosomes in cancer tissue behaved somewhat differently from those of normal tissue: the chromosomal mechanism functioned less accurately, mitotic deviations of all kinds being frequent. Ever since the end of the last century, etiological connections have been suggested between chromosomal aberrations and the origin of malignancy. Names such as von Hansemann and Boveri are associated with this idea. is the first modern cytogeneticist to stress the tumor tissue as a genetically heterogeneous cell population and to visualize the origin and growth of tumors as a result of selective processes among the constituents of this population. He based his ideas on careful and extensive chromosome studies in plant and animal tumors. The followiiig quotation out of Winge’s 1930 paper2 (p. 727) illustrates his view of the origin and growth of tumors: “Ich betrachte den malignen Tumor als bestehend aus Abkommlingszellen von einer oder mehreren Zellen, die durch eine lokale Reizung einen abnormen Chromosomenbestand bekommen haben, in dem besonders haufig ein Verlust verschiedener Chromosomen und eventuell Verdoppelungen ariderer Chromosomen eingetreten ist, was eine vergrosserte Wachstumsintensitat und im iibrigen cinen gestohrten Metabolismus mit sich fiihren kann.” This quotation shows that Winge was well aware of the stemliiie concept. Through genetic variation in the tumor, manifested as asymmetric or multipolar mitoses, the basis for an increase ic virulence is available: “Es geschieht wahrend des Vordringens des malignen Gewebes einc Selektion unter den aberranteii Zellen” (p. 728). Winge considers chromosome doublings as especially significant for the development of malignancy. In his analysis of tar carcinomas of micc, he observed that the most malignant neoplasms usually had hypotetraploid chromosome numbers, while precancerous stages still maintained near-diploid numbers. Winge’s view of the cancer tissue as genetically a mosaic, in which the most viable cell lineages are seIected to propagate the tumor, is still largely valid. On the other hand, it does not seem to follow necessarily that the original, primary induction of malignancy is caused by the same factors as those responsible for changes in the established tumors, as evidently assumed by Winge. T t is one of the most urgent tasks for chromosome research in cancer to focus on the early stages of cancer growth, or even on precancerous stages. The discovery during recent years that ascites tumors permit a much more Winge’p

Tumor etiology and chromosome pattern.

Fibrosarcomas induced in Chinese hamsters and rats by Rous sarcomla virus and 7,12-dimethylbenz(a)anthracene are associated with nonrandom chromosome variation. Although histologically indistinguishable, the tumors induced by the virus or chemical in each host species are characterized by completely different karyotypic patterns.

Inverse relationship between chromosome ploidy and host-specificity of sixteen transplantable tumors☆

Abstract A representative series of four lymphomas, five carcinomas, one melanoma, and six sarcomas (largely in the ascites form) has been surveyed for chromosome number distribution, growth capacity in foreign strains and, where possible, histocompatibility genes. This comparative analysis, supported by relevant data from other laboratories, has demonstrated a consistent influence of ploidy (2n versus 4n) upon degree of antigenicity. The diploid neoplasms were entirely strain-specific and always regressed in unrelated lines. The tumors characterized by a tetraploid modal value were able to grow in and kill hosts of one or more foreign genotypes, or even showed complete genetic indifference. This inverse relationship between chromosome number and host-requirements appears to be independent of the tumors histology, virulence, strain of origin, method of induction, and transplantation history. The findings are suggestive of “immunologic selection” from a relatively narrow (mode near 2n) or wide (mode near 4n) range of viable antigenic variants in a malignant cell population.

Measles associated chromosome breakage

I n 1962 we repor ted chromosome breaks in pe r iphera l whi te blood cells (1) of pa t i en t s wi th cl inical measles (rubeola). This work was pa r t of a s t u d y on the re la t ionship be tween viruses, chromosomes, and carcinogenesis. The f i rs t sys tem s tud ied in this p ro jec t was a t u m o r virus, the S c h m i d t R u p p i n s t ra in of the Rous sa rcoma virus. This produces tumors no t only in the chicken and fowl bu t also in a wide va r i e t y of m a m m a l s (2). W h e n this v i rus was s tud ied in the r a t i t was no ted t h a t chromosome b reakage occurred when the t u m o r was s tud ied in vivo or in t issue cul ture and also when the virus was a d d e d to no rma l r a t cells in t issue cul ture (3). When the chromosomes of the r a t t umors induced b y this v i rus were s tud ied in serial t r ansp lan ta t ion , new chromosome types were seen to arise. This could only occur as the resul t of previous b reakage and reunion or heal ing be tween the broken ends of chromosomes. W h e n r a t tumors of 0 passage were p u t into t issue cul ture and ser ia l ly t ransfer red , t h e y underwent a cel lular t r ans fo rma t ion wi th change in morpho logy and g rowth character is t ics . A t the t ime these cellular changes took place, large numbers of chromosome breaks and rea r rangements were seen. Af te r this, the cells s tab i l ized and behaved as a cell line. The breaks and rea r rangements had no t been seen in p repara t ions made before the morphologic t r ans fo rma t ion and have no t been seen since t h a t t ime. F ina l ly , when cell-free virus was added to d iploid r a t embryo cells in t issue cul ture and the f irst and second divisions af ter the add i t ion were s tud ied ehromosomal ly , i t was found t ha t there was an increase in the number of b reaks over the controls. On the basis of these observat ions i t was fel t t h a t b reakage was p lay ing a role in the in te rac t ion of the cell and t u m o r virus.

CHROMOSOME ABNORMALITIES IN VITRO IN HUMAN LEUKOCYTES ASSOCIATED WITH SCHMIDT-RUPPIN ROUS SARCOMA VIRUS.

Chromosome breakage was observed in human leukocytes in vitro when Schmidt-Ruppin strain of Rous sarcoma virus was added to the cultures. Similar additions of Bryan strain of Rous sarcoma virus had no such effect.

The stemline chromosomes of three cell lines representing different vertebrate classes

The chromosomes of the stemlines were analyzed in three cell lines stored in the Cell Bank of the South Jersey Medical Research Foundation, Camden, N. J. The cell lines were of marsupial, amphibian and teleostean origin, and 10 to 25 complete chromosome analyses in each of them formed the basis for idiograms. In the marsupial and the frog, comparison could be made with the normal karyotype of the species.

Alternative involvement of two cytogenetically distinguishable breakpoints on chromosome 8 in burkitt's lymphoma associated translocations

The chromosomes of 16 Burkitts lymphoma (BL) derived cell lines were submitted to high-resolution G-band analysis. They included seven lines with t(8;14), three with t(2;8), and four with t(8;22). The translocation breakpoint in chromosome #14 was located in 14q32.3, in chromosome #2 in 2p11.1, and in chromosome #22 in 22q12.12. In chromosome #8, the translocation breakpoint was located in two cytogenetically distinct subbands: 8q24.1 in cell lines with t(8;14) and t(2;8) and 8q24.22 in cell lines with t(8;22). In the light of recent molecular findings, these results indicate that the distance between the c-myc gene, located in 8q24, and the Ig sequences might be much larger in BL lines with t(8;22) than in those with t(2;8).

Cytogenetic findings in 111 ovarian cancer patients: Therapy-related chromosome aberrations and heterochromatic variants☆

The chromosomes of 111 ovarian cancer patients were studied in G- and C-banded slides from peripheral blood lymphocyte (PBL) cultures for chromosome damage caused by chemotherapy and radiotherapy and for asymmetry of the constitutive heterochromatin of chromosomes 1, 9, and 16. We also monitored the survival of these patients to determine whether any secondary neoplasia induced by the therapy and report the findings of our investigations. Melphalan (MEL) was the only drug used in single-drug chemotherapy. The incidence of chromosome abnormalities in melphalan-treated cells (25%) was higher than in the control group (17%). The incidence of structural changes was also higher (10.5%) in the MEL-treated group than in controls (6%). After treatments with combinations of drugs, the incidence of structural changes remained at the same level (11%). In the patients receiving combined treatment with MEL and radiation, the rate of structural changes increased dramatically (24%). The overall rate of chromosome aberrations in this group was also higher (50%). Combination of two or more drugs and radiation produced only 14% structural chromosome changes. The overall rate of chromosome aberrations was also low (20%) in this group. Of 111 patients studied, only 33 were alive 6 years after initiation of the study. Of the surviving patients, eight had rearranged chromosomes in the first analysis. After 5 years, new blood samples were collected from these patients and chromosome analyses showed abnormal karyotypes in all eight patients. All chromosome abnormalities in the second analysis were completely unrelated to those in the first analysis, however. Whether the chromosome changes in the second analysis were due to therapy or to other unknown factors could not be determined. Data on C-banding and the distribution of inversions indicated that 91% of the patients had C-band heteromorphisms of chromosomes 1, 91% had heteromorphisms of chromosome 9, and 69% had heteromorphisms of chromosome 16. Furthermore, inversions were observed in chromosome 1 (41% of patients), chromosome 9 (28% of patients), and chromosome 16 (5% of patients).

Novel cytogenetic expression of gene amplification in actinomycin D-resistant somatic cell hybrids: transfer of resistance by centric chromatin bodies.

SEWATC13 mouse cells, resistant to 0.1 μg/ml of actinomycin D (AMD), were fused to AMD-sensitive cells of the Chinese hamster ovary cell line (CHO). Twentytwo hybrid clones were isolated and put into serial culture in the selective medium. Unexpectedly, identifiable mouse chromosomes were found only in one of the hybrids. All the others had only hamster chromosomes and, in addition, numerous chromatin bodies (CBs), mostly small and irregularly shaped, but also larger, more chromosome-like ones. The CBs were distinctly C-band positive and a mouse satellite probe hybridized strongly to them. The AMD resistance of the murine parental cells had previously been attributed to gene amplification in two large homogeneously staining regions (HSR-AMD1 and 2). They were not observed in the hybrid cells but had supposedly reappeared in the guise of the CBs. It was established by Southern DNA blot analysis that amplified DNA sequences, localized to the HSRAMD1 and 2 of the SEWA parent were present in multiple copies in the hybrids. It was also established by in situ hybridization that they were located in the CBs. Unlike double minutes (DMs) the CBs were all centric.