Experimental Models for the Study of Carcinogenesis

A number of models for the study of carcino-genesis have been developed over the years. Historically, two of the most useful ones have been the initiation-promotion model of mouse skin carcinogenesis (the "skin-painting" model) and the induction of liver cancers in rats.

The classic model of carcinogenesis is the single application of an initiating agent such as a polycyclic aromatic hydrocarbon followed by the continuous application of a promoting agent like TPA to the backs of shaved mice. Much of what we know about tumor initiation, promotion, and progression has come from this model system.

Initiation and promotion during mouse skin carcinogenesis produce multiple benign squamous papillomas. A few squamous cell carcinomas eventually arise from the papillomas over many months. However, malignant conversion can be speeded up by exposure of papilloma-bearing mice to mutagens, which activates oncogenes such as H-ra.s and causes loss of tumor suppressor genes such as p53, as noted above.

The mouse skin carcinogenesis model is also a useful one in which to study the role of diet and chemopreventive agents in carcinogenesis (see also Chapter 9). For example, calorie-restricted diets have been shown to reduce the number and size of papillomas during and following promotion with TPA in DMBA-initiated SEN-CAR mice.85 Furthermore, the latency period for occurrence of carcinomas was increased and the total number of carcinomas was decreased. Application of apigenin, aplant alkaloid,86retinoic acid,87 and prostratin, a nonpromoting phorbol ester88 have been shown to inhibit the promotion phase (appearance of papillomas) of mouse skin carcinogenesis.

Multistage carcinogenesis has also been observed for liver tissue. For example, Peraino et al.89 observed that a 3-week exposure of rats to AAF in the diet produced only a small number of hepatomas after several months, but if the animals were subsequently treated with phenobarbital for several months after carcinogen feeding was discontinued, a high incidence of hepatomas was noted. Similar results have been obtained by Kitagawa et al.,90 who fed rats a nonhepatocarcinogenic dose of 2-methyl-N,N-dimethyl-4-aminoazobenzene for 2 to 6 weeks, and then a dietary administration of phenobarbital for 70 weeks. By 72 weeks, many large hepatocellular carcinomas had developed in the phenobarbital-treated animals, whereas only a few small tumor nodules were observed in the rats not given phenobarbital. Thus, the action of phenobarbital appears to be analogous to that of TPA in the mouse skin system—that is, it "fixes" the damage to cells induced by an initiating agent and causes a clone of cells arising from a damaged cell to proliferate. However, whereas TPA stimulates DNA synthesis and hyperplasia in skin, phenobarbital produces only a transient and relatively small increase in DNA synthesis in liver. Perhaps that is all that is needed to fix the carcinogenic damage and to allow for the initial proliferation of a damaged clone of cells. Once the damaged clone is present, it could undergo alteration due to its genetic instability and gradually progress to a detectable malignant tumor. This idea is supported by the experiments of Pitot et al.,91 who treated rats with a single dose of diethylnitrosamine by intubation 24 hours after partial hepatectomy (partial removal of the liver), which stimulates DNA synthesis and cell proliferation in the remaining tissue. If the animals were then treated, starting 8 weeks later, with phenobarbital in the diet for 6 months, many small, phenotypically heterogeneous foci characterized by glucose-6-phosphatase-deficient areas, ATPase-deficient areas, and g-glutamyltranspeptidase-containing areas developed in the liver. Many of these animals also had hepatomas, for which the enzyme-altered foci appear to represent the early stage of neoplastic development. Thus in this case, phenobarbital appears to have stimulated the replication of dormant initiated cells, which, in the absence of the promoter, would not have proliferated. If each enzyme-altered focus observed in these experiments were a clone derived from a single cell, about 104 to 105 cells in the liver were "initiated" by diethylni-trosamine, and a very small number of these subsequently underwent clonal proliferation during phenobarbital feeding.91 Thus the conversion of these abnormal foci, or early nodules, as they have been called, to a malignant neoplasm is a rare event.

Newer models of carcinogenicity have involved the use of knock-out or knock-in rodent models, in which various oncogenes, tumor-suppressor genes, or susceptibility genes have been engineered into or out of rodent embryos (usually mice). This process has enabled the definition of some of the genes that are key to various steps in the tumor-initiation promotion and progression steps. These tumor models are now being super-ceded by conditional genetic knock-out models in mice that allow for the controlled expression of oncogenes or tumor suppressor genes in a way that more closely mimics "spontaneously" arising human cancers (Table 2-2).

Conditional gene expression in the mouse has been achieved by mutations induced by FLP/ FRT or Cre/lox P site-specific recombination

Table 2-2. Conditional and Inducible Mouse Tumor Models

Tumor Type Conditional or Inducible Gene*

TUMOR MODELS THAT USE CRE/IOXP OR FLP/FRT RECOMBINATION SYSTEMS

Colorectal adenomas ApcIoxF

Mammary adenocarcinomas Brca1IoxF + Trp53+/~

Mammary adenocarcinomas Brca2IoxF + Trp53IoxF

Mammary adenocarcinomas Brca2IoxF

Schwannomas Nf2IoxF

Lung adenocarcinomas StopIaxF +KrasG12D

Pituitary tumours Rb IoxF or RbFRT

Medulloblastomas RbIoxF + Trp53IoxF

Liver haemangiomas VhlIoxF

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