450 GENETICS OF SOMATIC CELLS 



As discussed in previous sections, it is also possible to establish, by techniques of 

 selective transfer, parallel sublines from the same original tumor differing in chromoso- 

 mal characteristics, 530, 531, 690 in isoantigenicity, 531, 717, 724 in the capacity to grow in 

 the ascitic form, and with regard to correlated phenomena of invasiveness and ability 

 to metastasize, 721, 1031, 1057 in hormone production, 412, 413, 414 and in drug resist- 

 ance. 761 • 762 Differences between such specific pairs may reveal more about underlying 

 mechanisms than comparisons between unrelated populations of normal and tumor 

 cells. 



TISSUE CULTURE 



The recent spectacular advances in tissue culture leading to the development of 

 cloning techniques, partially or fully defined media, suspension cultures, and highly 

 improved procedures for chromosomal studies, bring in-vitro methods into the fore- 

 front of interest in every discussion on somatic-cell genetics. It would be impossible 

 to review these developments here in detail; consideration will be restricted to a brief 

 survey of available marker characteristics and discussion of the relationships between 

 systems in vivo and in vitro. 



Marker characteristics. — The conscious, large-scale selection of cellular variants 

 differing in phenotypic marker characteristics has been mainly initiated by the de- 

 velopment of cloning techniques. The growth of single, isolated cells in vitro was first 

 achieved by Sanford, Earle, and Likely. 1150 Convenient, large-scale procedures 

 were developed by Puck and associates, 1025 and various new methods or modifications 

 were introduced by others with the purpose of combining the ease and large-scale 

 operation which is characteristic of Puck's technique with the rigorously critical assur- 

 ance of single-cell origin inherent in the method of Sanford et a/. 35, 449 The markers 

 studied include resistance to drugs, viruses, and radiation and nutritional, morphologic, 

 and chromosomal characteristics. 



Puck and associates 1025 have isolated clones ofHeLa cells differing from the majority 

 of the population with regard to their appearance, requirements for human serum, or 

 resistance to Newcastle-disease virus. As an interesting example, the case of lines 

 S\ and S3 may be quoted. When the standard medium was supplemented with 3 

 per cent human serum only, the plating efficiency of S3 was 100 per cent and that of 

 S\ was zero. The S\ cells displayed a cloning efficiency of 100 per cent, however, il 

 plated on a feeder layer of irradiated S3 cells metabolically active but incapable of 

 further multiplication. The behavior of these lines was stable during continuous 

 passage. Other types of stable variants have been produced by X irradiation oiHeLa- 

 cell populations. 1025 Some of these were morphologic variants, throwing off bizarre 

 cells, and enlarged monstrosities, and this behavior was bred true during prolonged 

 passage. Other X-ray-induced variants were nutritionally different from the original 

 type. With regard to spontaneous or X-ray-induced variants that were recognizable 

 by a difference in their clonal appearance, Puck stressed particularly the importance 



