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dcyphr | v-ras genes from harvey and BALB murine sarcoma viruses can act as initiators of two-stage mouse skin carcinogenesis.

Summary


The retrovirus HaMSV was used to introduce activated ras genes to mice. After the tumor promoter TPA was added, the same mice developed benign tumors. Some benign tumors eventually became malignant. Cells initiated by HaMSV can remain in the body for at least four months. This study shows that activating ras genes in mice has the same effect as giving them chemical carcinogens. This suggests that ras and other oncogenes have an important role in tumor development.


Introduction


Cancer development is now recognized as a multi-stage process. The progression from stage to stage may be spurred on by the activation of proto-oncogenes. These genes have remained in the genome because they support cell growth. Scientists know that they’re involved in tumor development because they’re activated in many cancers. However, at the time this article was written, the stage of tumor development during which these genes activate was unknown. Their activation’s exact effect on cells was also not well understood. 


To uncover these unknowns, the authors of this article made tumors form in mice. They did this by giving them some amount of a carcinogen. They then gave the mice chemicals that made visible tumors appear. These chemicals are called promoting agents. The tumors that appeared afterwards were usually benign, but some go through more change and eventually invade other parts of the body.


During one round of these experiments, the authors found that the Harvey-ras gene, abbreviated as c-rasH, was frequently activated at the same time benign tumors began to form. This suggested that the gene is activated pretty early in the tumor development process. If this is the case, one would expect that the activation of this gene would achieve the same effect as exposing a mouse to a carcinogen. From then, adding promoting agents to mice with activated c-rasH genes would likely make benign tumors form, just as if a carcinogen was added first. This is what the authors show with the study detailed in this article.


Results


Induction of Skin Papillomas Using HaMSV and TPA


In the experiment described, the authors initiated tumor development in mice by exposing them to the HaMSV retrovirus, which is expected to activate the c-rasH gene. Afterwards, some of these mice were treated with TPA while some were treated with acetone. The mice that received acetone did not develop any tumors. The mice that received TPA developed visible tumors in 4-5 weeks. When compared to the tumors initiated by chemical carcinogens, the tumors initiated by HaMSV grew faster but looked similar. Tumors initiated by HaMSV also had cells that were less differentiated.


HaMSV-Initiated Cells Can Remain Latent in Epidermis for Several Months  


Cells initiated with chemical carcinogens are able to stay in the body for up to a year. The authors tried to see if the same is true for cells initiated with HaMSV. HaMSV-exposed mice that received promoting agents four months after infection developed tumors, showing that HaMSV-initiated cells can stay in the body for extended periods of time. However, these tumors were smaller than the ones developed in mice that received promoting agents right after infection. This suggests that some initiated cells in HaMSV-infected mice can be lost. 


Conversion of HaMSV Papillomas to Carcinomas


The results of the study suggest that benign tumors developed from HaMSV-initiated mice spent less time dormant before becoming malignant when compared to chemical-initiated mice. The study also seems to suggest that a greater percent of HaMSV-initiated benign tumors eventually become malignant than chemical-initiated tumors. However, this study should be repeated with more mice to properly support these conclusions.


Clonality of HaMSV-Induced Skin Tumors 


Through restriction enzyme digests, the authors concluded that the benign tumors that appeared in HaMSV-initiated mice contain cells that came from several different populations. However, the malignant tumors that formed in these mice were likely caused by an event that took place in one or two cells.


Expression of v-rasH Sequences in HaMSV Tumors 


The authors wished to make sure that HaMSV-initiated mice did indeed have more activated rasHgenes than chemical-initiated mice. A northern blot revealed that a chemically-initiated benign tumor had only normal copies of the rasH gene. It also showed that there was greater expression of rasHin HaMSV-initiated tumors than in chemical-initiated tumors. 


Expression and Cytochemical Localization of The rasH gene of HaMSV differs from the equivalent norv-rasH P21 in HaMSV-Tumors


HaSMV copies of the rasH gene differ normal copies of the rasH gene, as they contain two mutations. These mutations alter the protein product they produce and endow it with different properties.


Discussion


One issue in uncovering the role of oncogenes in tumor development was whether oncogene activation was the cause or result of tumor development. The study described in this article shows that oncogene activation could be a key step in cancer formation. This is evidenced by the fact that activation of an oncogene imitates the effect of chemical carcinogen exposure in mice.


ras Gene Mutation As the initiation Event in Mouse Skin Carcinogenesis 


These experiments can’t completely validate the idea that chemical carcinogens must directly cause the c-rasH gene to mutate in order for mouse skin carcinoma to form. Nor can they tell whether its mutation occurs right at the start of or after tumor initiation. However, they can strongly support the idea that c-rasH mutation is the initiating event of this cancer.


Chemical and Viral Initiation: Similarities and Differences


This section summarizes and elaborates upon the differences between tumors initiated with chemical carcinogens and those initiated with retroviruses. Virus-initiated tumors grow faster. Virus-initiated cells are dormant for less time before they become benign tumors. Virus-initiated mice tend to have more activated rasH genes.


Synergism between ras Gene Activation and TPA 


The activation of the ras gene alone isn’t enough to trigger tumor development. It requires TPA in order to manifest into a tumor. In cases where the ras gene was activated and TPA wasn’t received, no tumors formed. This implies an interaction between TPA and the ras protein product that is necessary for tumor development.


Conclusions and Prospects


The authors have shown that v-ras genes can be introduced in live animals and stimulate skin carcinoma development. Prior to this study, it was believed that HaMSV was only able to induce sarcomas or erythroid leukemias. Future studies could look into the potential for other oncogenes to cause cancers after being introduced through a retrovirus.


Experimental Procedures


The authors go over the various techniques used to carry out the study, including cell culture, virus preparation, DNA isolation, and so on.