29 May

Because of the relative homogeneity of transformed cell populations in vitro, it has been possi- ble to make direct comparisons between both messenger RNA and protein populations of trans- formed cells with those of their nontransformed  counterparts.  In these instances,  it becomes extremely important to be rigorous in the populations being compared, as noted above.

Messenger RNAs in Normal and Transformed Cells in Culture

Comparative  studies of different RNA populations in normal and transformed cells have been somewhat cyclic with respect to methodologies. As an example, in 1980, Moyzis and associates compared nuclear RNA and polysomal poly(adenylic acid) messenger RNA in normal SHE cells and a cell line that had originally been treated with benzo[a]pyrene. Utilizing the liquid hybrid- ization techniques popular at that time, they were able to demonstrate relatively few changes in the qualitative pattern of gene expression in these two cell populations. However, when specific messenger RNAs were investigated after transformation by a variety of methods, distinct quanti- tative changes could be found between normal and transformed cells in culture. Some examples of such instances are seen in Table 16.5. While many more examples might be given, many of them do not satisfy the criteria discussed at the beginning of this chapter. However, the examples given do indicate dramatic differences in the messenger RNA levels of the specific genes indi- cated. Most of the examples are matrix or cytoskeletal proteins, with one oncogene-related  ex- ample. There are obviously numerous other examples that could be given in the oncogene area, but that would only further emphasize the point made in the table.

Within the last few years, a variety of technologies  have been developed for examining large numbers of messenger RNAs in cells, both in vivo and in vitro. Because of the ease and “purity” of the use of cells in culture, many of the examples of these technologies are in cultured cells. Table 16.6 lists some of the more modern technologies that have been utilized, with appro- priate references. The student is urged to examine the references for details of the methods them- selves.  Of  the  methods  listed,  the  differential  display  method  is relatively  simple  and

Table 16.5 Expression of Some Specific mRNAs in Transformed Compared with Their Nontransformed  Controls

inexpensive but exhibits difficulties from false positives and negatives. Subtractive hybridization is an elegant technology that requires some experience to obtain appropriate results. The DNA microarray methods presently in use allow for the examination of the expression of 104 or more genes but are extremely expensive. The less expensive “gene screens” usually have fewer than 103 genes with which to examine differential expression from different mRNA populations. As an example, the subtractive hybridization method has been utilized to compare a normal strain of hu- man mammary cells with an aneuploid neoplastic cell line obtained from a pleural effusion (Lee et al., 1991). Although the comparison here does not conform to the criteria indicated at the begin- ning of this chapter, it does demonstrate that the method allows for the identification of differential expression  of specific genes in two populations  as well as the identification  of genes not yet described.

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