Loss of contact inhibition of growth and immortalization of primary cells

Immortalized B lymphocytes are but one example of cells available in the laboratory that have properties intermediate between the two extremes of continuous cell lines and primary cells. These cell types have undergone transformation and have at least some of the properties of tumor cells. Culturing primary cells for long periods can generate transformed cells. During the time in culture, there is a random accumulation of mutations that alter a critical number of growth control genes encoded by the cell. At a critical point during cultivation (the actual point will depend upon the cell type—it is usually 12—15 generations with fibroblasts) cells suffer from the cumulative effects of aging (senescence) where nearly all enter a crisis period and undergo apoptosis and die.

Senescence is a consequence of the defective replication of chromosomal DNA, which is linear, and (as discussed in Chapter 13) cannot completely replicate itself at the ends. Thus, each round of DNA replication results in the loss of the critical telomeric sequences at the end of chromosomes. Normally, the telomeres bind to a number of specific cellular proteins, which mask the chromosomal ends that are structurally equivalent to double-stranded DNA breaks. When the ends are unmasked, a number of important cellular defenses are activated that initiate the apoptotic pathway.

As will be outlined in the discussion of carcinogenesis in Part V, a major factor in carcinogenesis is the abrogation of the normal apoptotic response to double-stranded DNA breaks. These breaks, if not properly repaired, can be mutagenic and can alter the function of numerous growth control and developmental genes leading to uncontrolled cell growth. In cultured cells undergoing crisis, a very few may be able to protect the ends of critical chromosomes by a combination of inappropriate recombination events along with the activation of the telom-erase enzyme normally active in early development that regenerates telomeric sequences lost during chromosome replication. Thus, cells surviving crisis share certain features of tumor cells. These immortalized cells become predominant and relatively rapidly overgrow the culture. Such cells eventually can be used to generate continuous lines.

Cells with the properties of transformed cells also can be isolated from tumors in an animal. Different tumor cells in an animal display one or several of the same transformation levels from normal cells that can be observed with the culture of primary cells. This is an important clue to the nature of the cellular events leading to cancers. It is important to be aware, however, that different tumor cells can display widely different deviations from normal growth properties of the cells from which they derive. Some tumor cells, especially those isolated early in the course of cancer development, display very few differences from normal cells — perhaps only the loss of contact inhibition of growth. Others, especially long after the cancer occurred, have many additional changes.

The process of change from primary cells to continuous line cells and the relationship between these cells and tumors in the animal of origin is shown in schematic form in Fig. 10.2. This process of change is a convincing experimental demonstration that the cellular changes in an organism from normal to cancerous involve multiple steps. The changes multiply as mutations of specific growth control and regulatory genes in the cells alter cell function and the cell's ability to respond to normal signals in the animal, limiting cell growth and function.

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Primary cells can be transplanted back into donor.

Further changes in cellular genes lead to a continuous line cell.

Primary cells can be transplanted back into donor.

Growth transformed cells can be isolated and cultured from tumor or mass, can cause same tumor when transplanted into syngenic animal.

Further changes in cellular genes lead to a continuous line cell.

Fig. 10.2 The progression of cells in culture from primary to transformed to continuous lines, and their relationship to tissues in the originating animal.

While it is not uncommon to generate an immortalized cell line by lengthy passage or other mutagenic processes, it is important to be aware that many tumor cells and some transformed cells that have lost contact inhibition of growth still have a finite lifetime. The genes controlling life span and response to contact inhibition are not identical and can be mutated together or separately.

One of the most fruitful aspects of the study of some viruses is that they can cause transformation of normal primary cells into cancer or tumor cells. Such virus-transformed cells, when reintroduced into animals, can cause tumors. Since this transformation requires a specific interaction between viral and cellular genes or gene products, the study of the process has led to much current understanding of carcinogenesis and the nature of cancer cells.

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