biochemical and genetic studies on mammalian cells

IN ~ITRO Vol. 7, No. 3, 1971

BIOCHEMICAL AND GENETIC STUDIES ON MAMMALIAN CELLS*~-

THEODORE T. PUCK+ +

Department o] Biophysics and Genetics, University o] Colorado Medical Center, Denver, Colorado 80220

Mammalian tissue culture began at the turn of the century. From the first it was recognized as a technique with an enormous potential, since it permitted study of cellular behavior under conditions of much greater control of the exter- nal environment than is possible inside the body. During the first half century of its development, progress was not spectacular. However, this pe- riod furnished the necessary tools which have now produced an explosive development. This Conference is reviewing some of the most re- cent achievements which are making in vitro studies of mammalian cells the most exciting field in modern science. The dedication of the new W. Alton Jones Cell Science Center of the Tissue Culture Association could hardly occur at a more opportune time.

In this paper will be listed some results of studies which originated in our laboratories at the University of Colorado or in which we have participated. The underlying aim of the mammalian cell laboratory of the Department of Biophysics and Genetics of the University of Colorado was to develop genetic-biochemical methodologies that would permit study of mammalian cells by the methods of molecular biology as applied to microorganisms. When this ap- proach was successful, an additional objective of understanding the nature of the cancerous process was added, and the creation of the Eleanor Roosevelt Institute for Cancer Research has enormously accelerated the laboratory's progress. The studies that are summarized here have

* From the Eleanor Roosevelt Institute for Can- cer Research and the Department of Biophysics and Genetics (No. 473). This investigation was aided by United States Public Health Service Grant 5 P01 HD02080 from the National Institute of Child Health and Human Development and by American Cancer Society Grant E-642A.

t Presented in the Symposium on Regulation in Tumor Cells at the Twenty-second Annual Meet- ing of the Tissue Culture Association.

++American Cancer Society Research Professor.

been carried out collaboratively by the following co-workers and the author: Dr. Lawrence Chasin, Dr. S. J. Cieciura, Dr. David Cox, Dr. Harold Fisher, Dr. Richard Ham, Dr. Abraham Hsie, Dr. Robert Johnson, Dr. Carol Jones, Dr. F. T. Kao, Dr. Philip Marcus, Dr. M. Oda, Dr. D. Petersen, Dr. Potu Rao, Dr. Arthur Robin- son, Dr. Gordon Sato, Dr. J. Steffen, Dr. J. H. Tjio, Dr. R. A. Tobey, Mr. Charles Waldren, and Dr. M. Yamada.

In the early phases of this program, techniques were developed which would permit relia- ble establishment of long term cultures from biopsies taken from any individual (1). The development of a method by which single cells from these cultures could be grown rapidly and reproducibly into discrete colonies made possible convenient isolation of mutant clones which furnished means for illuminating genetic processes (2). The identification and classification of human chromosomes and the demonstration of their involvement in a large group of human diseases have opened up a new and exciting chapter of medicine in which both fundamental and clinical discoveries continue to appear rapidly and consistently (3, 4).

In an effort to make definitive studies on single genes, the molecular nutritional requirements of the Chinese hamster cell were delineated so that auxotrophic mutations could be produced (5). A method for recognition and isolation of such mutants was developed, in which a large cell population is treated with bromodeoxyuridine (BUdR) in a minimal medium that will support the growth only of the prototrophic cells (6). These multiply and incorporate the drug into the DNA, while the auxotrophs remain dormant. The culture is then illuminated with visible light. Incorporation of BUdR into the DNA shifts its absorption spectrum toward the visible region, so that the overwhelming majority of the prototrophic cells is destroyed, leaving the auxo-

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trophs largely unaffected. An enriched medium is then substituted and colonies are formed by the auxotrophic cells. These colonies then can be picked out, and their nutritional requirements determined.

Spontaneous appearance of auxotrophic mutants in mammalian cells was found to be extremely small, usually less than 10 -~ per generation. However, when cells were treated with physical and chemical mutagenic agents like X- irradiation or ethylmethanesulfonate, large numbers of stable mutants were isolated (7). A par- tially monosomic Chinese hamster cell was used for these experiments, and evidence was secured to show that at least some of the mutants obtained represent gene changes that lie in the monosomic part of the karyotype. Methods have been devised which promise to introduce further monosomies into such cells, and these should ex- tend the variety of mutants available for genetic biochemical study. Approximately 10 different mutants have now been isolated. The biochemis- t ry of the blocks is under study and has been delineated in some eases (8, 9). The existence of this group of mutants has made possible a variety of interesting genetic experiments.

These mutants lend themselves particularly well to complementation analysis by means of the techniques for cell fusion which were developed in other laboratories. If two mutants are hybridized either by spontaneous fusion or through the use of ultraviolet-irradiated Sendai virus, the heterozygote can be tested for its ability to grow in the absence of both nutritional deficiencies (10). Thus, fusion between a glycine-deficient and a hypoxanthine-deficient mutant makes possible determination of whether these deficiencies are dominant or recessive. In the latter case, the hybrid cell will form stable colonies in the absence of both glycine and hypoxanthine, whereas in the former case the dominant deficiency would still be displayed by the heterozygote. To date, all of the mutations which we have produced have proved to be recessive by means of this test, and the presump- tion is therefore that the mutations involve structural rather than regulatory genes.

Even more critical analysis is possible if one uses, for such an experiment, two mutants which require the same metabolite for growth. In that case, one can determine whether the two mutants are blocked in the same gene or in different genes (8, 9). In the former case, the hybrid

cell will still require the metabolite in question, whereas in the latter, the nutritional deficiency will have been removed. In one series of experiments, 13 different glycine-requiring mutants, produced by a variety of different mutagenic agents, were fused two at a time, and the hybrids were examined for a glycine requirement. The series of mutants was found to form four separate complementation groups such that hybrid formation between two members of the same group would not remove the glycine deficiency, whereas hybrids formed from cells in different groups no longer required glycine. One of these mutant classes lacks the serine hydroxy- methylase enzyme which converts serine to glycine. The other three have blocks connected with various stages of tetrahydrofolate metabolism. This methodology appears to be directly applica- ble to patients with hmnan genetic diseases, in order to determine whether or not patients with the same clinical picture suffer from a mutation at the same genetic locus. Complementation analysis of hypoxanthine-requiring mutants has also been recently carried out.

These methodologies have made possible quantitative examination and comparison of the action of mutagenic agents on mammalian cells (11). In the course of these studies, the action of each agent was examined for its ability to produce specific gene mutations and to produce chromosomal aberrations. In this way a more complete picture of the action of the compound is possible than if any one action were studied. Moreover, certain simplifying characteristics of the action of certain agents have been uncov- ered.

The first step was to determine the single cell survival curve for each compound studied. I t was found that whereas some agents yielded one-hit curves and others multihit responses (i.e. the curve exhibited an initial shoulder) all the curves obeyed a simple target theory model. As is conventional in quantitative microbiology, Do, the mean lethal dose, was taken as the dose needed to reduce the number of survivors to the fraction 1/e = 37% as determined in the region where the curve is flowing in a simple exponential fashion. The mutagenic deficiency and the chromosome breakage efficiency were then expressed in terms of the Do value for each of the agents tested.

Such studies showed that all of the physical and chemical agents so far tested fall into three

MAMMALIAN CELL STUDIES 117

different groups. One group, exemplified by caffeine, produces ehromatid breaks but no detecta- ble single gene mutations under the conditions of study. ICR-191, an acridine mustard, kindly furnished by Dr. H. J. Creech, Chemotherapy Di- vision, Institute for Cancer Research, Fhiladel- phia (11), which presumably produces frame- shift mutation, produces few or no chromosome aberrations but is a very effective single gene mutagen. Finally, methylating agents, X-rays, and ultraviolet light were found to produce both kinds of lesions in good yields. These methodologies appear to lend themselves to the screening of drugs, food additives, and environmental pol- lutants for their mutagenic action on mammalian cells.

I t has been possible by means of these studies also to examine the action of carcinogenic agents in this manner (12, 13). Such studies might be expected to illuminate the role of mutation in carcinogenesis. Four different carcinogenic nitroso-compounds were examined in detail. All of them were found to be highly active as single gene mutagens and as chromatid breakage agents. Their single cell survival curve exhibited extremely large variations from one another. Some were single hit while others had an extremely long shoulder, and values of Do obtained from such curves differed from each other con- siderably, the ratio of the largest to the smallest being 5 • 10'. Large differences were also found in the efficiency of single gene and chromosome breakage production. However, when these latter values were expressed in terms of the Do value for each compound, the resulting values for single gene deficiency for all four compounds were remarkably constant. A similar constancy was found for production of chromatid breaks. I t would appear, then, that the use of the Do value is a useful method for comparison of ac- tivities of different agents in studies of this kind (13).

Studies of this kind can also be used to eluci- date the mechanism of action of different agents. For example, caffeine converts the survival curve produced by X-rays and ultraviolet light from a multihit to a single hit curve. The Do value of the resulting curve, however, remains very close to that obtained in the absence of caffeine. For these reasons, and because of the evidence obtained in other laboratories of the nature of caffeine's actions on ultraviolet-irradiated cells, it seems reasonable to attribute the

action of caffeine to the inhibition of cellular repair processes which are capable of neutraliz- ing the effects of small but not large doses of irradiation. However, when the effect of caffeine is studied on the survival curve of nitrosodi- methylamine, which also normally exhibits a large shoulder, the shoulder is hardly affected at all. The presumptive conclusion then can be drawn that the shoulder in the survival of nitrosodi- methylamine is due to different processes than those that operate in the case of ultraviolet and X-irradiation. Life cycle analysis has revealed the further interesting fact that caffeine added to cells does not affect them in the first generation but strongly inhibits the multiplication cycle thereafter, as though caffeine is interfering with a process which normally produces a suffi- cient excess of metabolites for one, but not two, complete life cycles (14).

Mutational studies have also been carried out in which the reversion of cells from auxotrophy to prototrophy has been studied. A variety of different kinds of behavior has been produced. Some auxotrophic mutations produced by X-irradiation have never been observed to revert, either spontaneously, or under the action of other mutagens. Reversion of other auxotrophies can be readily accomplished by a variety of mutagenic agents. I t seems reasonable to expect that such studies will aid in identifying the specific kind of mutational event that has occurred in each case (15).

Other workers have shown that genes can be located on the chromosomes of mammalian cells, by taking advantage of the extensive chromosome loss which occurs in certain interspecific hybrids after cell fusion in vitro. When normal human cells have been fused with mouse cells which contain a specific genetic defect affecting growth, the hybrids, selected under conditions which fail to support the growth of either parental cell, are cultured until there is maximal loss of human chromosomes (16). Any human chromosome which is uniquely common to all the surviving clones is presumed to carry the normal gene which compensates for the defect in the mouse cell. The Chinese hamster cell, which we have been using in this laboratory, has turned out to be remarkably useful in experiments of this kind. For one thing the ease of production of specific gene mutations makes available a large number of loci for such linkage group determination. Even more important,

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however, may be the fact that loss of human chromosomes occurs much more rapidly and completely when Chinese hamster cells are used for this hybridization experiment. The rapidity of this process may in large part be determined by the disparity in the growth rate of the two parental cells. Presumably, this operates in two ways. First, since the two cells have different rates of traverse of their respective life cycles, asynchrony in the two chromosomal complements is to be expected in a large fraction of the initial fused population (17). Johnson and Rao (18), in this laboratory, have shown that a set of mitotic chromosomes in a hybrid cell can in- duce premature condensation in a set of inter- phase chromosomes. When the latter are in G1 or S, their condensation results in abnormal chromosomal structures, which are presumably lost in relatively large numbers. In addition, however, the generation time of the Chinese hamster cell is approximately half of that of the human cell. Cell hybrids grow slowly initially, approximating the growth rate of the more slowly reproducing parental cell. If extensive loss of chromosomes of the slower parent occurs, the hybrid approaches the growth rate of the faster parent. Therefore, an automatic selective advantage is established for rapid isolation of those hybrids which have lost the maximal number of human chromosomes. Studies are continu- ing, to assign the markers so far available to their respective linkage groups.

The Chinese hamster ovary cell has also proved to be useful in another kind of reaction. Treatment of these cells in vitro with dibutyryl adenosine cyclic 3 ' ,5 '-monophosphate converts the culture from one of compact, randomly ori- ented cells that grow in multilayers to a mono- layer of elongated fibroblast-like cells growing parallel to one another. Testosterone propionate, which has a similar though smaller effect at high concentrations, potentiates the action of dibutyryl cyclic AMP, even when added at very low concentrations. The transformation is recogniza- ble within 1 hr, affects cells throughout all or most of the life cycle, and is completely reversi- ble. Both cell forms reproduce with approximately the same generation time. Agents like colcemide and vinblastine, which inhibit assem- bly of microtubules, prevent the transformation to the fibroblast form. I t is postulated that the dibutyryl cyclic AMP and testosterone act by promoting organization of mierotubules from

protein monomers. This system appears useful in study of the regulation of phenotypic expres- sion in mammalian cells (19, 20).

The purpose of this paper has been to demon- strate the wealth of new kinds of experimental approaches which are offered by in vitro studies of mammalian cells. The next decade should see remarkable developments that can be expected to illuminate large areas of mammalian cell behavior.

REFERENCES

I. Puck, T. T., P. I. Marcus, and S. J. Cieciura. 1956. Clonal growth of mammalian cells in vitro. Growth characteristics of colonies from single HeLa cells with and without a "feeder" layer. J. Exp. Med. 103 �9 273-283.

2. Puck, T. T.. S. J. Cieciura, and A. Robinson. 1958. Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. J. Exp. Med. 108: 945-956.

3. Tjio, J. H., and T. T. Puck. 1958. The somatic chromosomes of man. Proc. Natl. Acad. Sci. U.S.A. 44 : 1229-1237.

4. Robinson, A., et al. 1960. A proposed standard system of nomenclature of human mitotic chromosomes. J.A.M.A. 174 : 159-162.

5. Ham, R. G. 1965. Clonal growth of mammalian cells in a chemically defined, synthetic medium. Proc. Nail. Acad. Sci. U.S.A. 53: 288- 293.

6. Puck, T. T., and F.-T. Kao. 1967. Genetics of somatic mammalian cells. V. Treatment with 5-bromodeoxyuridine and visible light for isolation of nutritionally deficient mutants. Proc. Natl. Acad. Sci. U.S.A. 58: 1227-1234.

7. Kao, F.-T., and T. T. Puck. 1968. Genetics of somatic mammalian cells. VII. Induction and isolation of nutritional mutants in Chinese hamster cells. Proc. Natl. Acad. Sci. U.S.A. 60: 1275-1281.

8. Kao, F.-T., and T. T. Puck. 1967. Genetics of somatic mammalian cells. IV. Properties of Chinese hamster cell mutants with respect to the requirement for proline. Genetics 55" 513-529.

9. Kao, F.-T., L. Chasin, and T. T. Puck. 1969. Genetics of somatic mammalian cells. X. Complementation analysis of glycine-requiring mutants. Proc. Natl. Acad. Sci. U.S.A. 64 : 1284-1291.

10. Kao, F.-T., R. T. Johnson, and T. T. Puck. 1969. Genetics of somatic mammalian cells. VIII. Complementation analysis on virus- fused Chinese hamster cells with nutritional markers. Science 164: 312-314.

11. Creech, H. J., E. Breuninger, R. F. Hankwitz, G. Polsky, and M. L. Wilson. 1960. Quanti- tative studies of the effects of nitrogen mustard analogs and other alkylating agents on ascites tumors in mice. Cancer Res. 20"

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[Suppl] Chemother. Screening Data VII. 471-494.

12. Kao, F.-T., and T. T. Puck. 1969. Genetics of somatic mammalian cells. IX. Quantitation of mutagenesis by physical and chemical agents. J. Cell. Physiol. 74: 245--258.

13. Kao, F.-T., and T. T. Puck. 1971. Genetics of somatic mammalian cells. XII. Mutagenesis by carcinogenic nitroso compounds. J. Cell. Physiol., in press.

14. Waldren, C., I. Rasko, and T. T. Puck. Data to be published.

15. Chasin, L., and T. T. Puck. Data to be published.

16. Weiss, M. C., and H. Green. 1967. Human mouse hybrid cell lines containing partial complements of human chromosomes and functioning human genes. Proc. Natl. Acad. Sci. U.S.A. 58: 1104-1111.

17. Kao, F.-T., and T. T. Puck. 1970. Genetics of somatic mammalian cells: linkage studies

with human-Chinese hamster cell hybrids. Nature (Lond.). 228: 329-332.

18. Johnson, R. T., and P. N. Rao. 1971. Nucleo- cytoplasmic interactions in the achievement of nuclear synchrony in DNA synthesis and mitosis in multinucleate cells. Biol. Rev. 46: 97-155.

19. Hsie, A. W., and T. T. Puck. 1971. Mammalian cell transformation in vitro. I. A morphologi- cal transformation of Chinese hamster cells produced by dibutyryl cyclic adenosine monophosphate and testosterone. Proc. Natl. Acad. Sci. U.S.A. 68: 358-361.

20. I-Isle, A. W., C. Jones, and T. T. Puck. 1971. Mammalian cell transformations in vitro. II. Further changes in differentiation state ac- companying the conversion of Chinese hamster cells to fibroblastic form by dibutyryl adenosine cyclic 3':5'-monophosphate and testosterone. Proc. Natl. Acad. Sci. U.S.A., in press.

biochemical and genetic studies on mammalian cells

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