Cell Transformation Detection

Cell Transformation Detection Assay

Chemicon (Millipore)

CHEMICON®'s Cell Transformation Detection Assay is an anchorage-independent growth assay in soft agar, which is considered the most stringent assay for detecting malignant transformation of cells. For this assay, cells (pre-treated with carcinogens or carcinogen inhibitors) are cultured with appropriate controls in soft agar medium for 21-28 days. Following this incubation period, formed colonies can either be analyzed morphologically using our cell stain solution or quantified with the cell quantification solution.

Neoplastic cell transformation results from the accumulation of genetic mutations that permit uncontrolled proliferation and independence from normal homeostatic regulation. Cell changes manifested by escape from control mechanisms, increased growth potential, alterations in cell surface, karyotypic abnormalities, morphological and biochemical deviations, and other attributes conferring the ability to invade, metastasize and kill.

Carcinogenesis is a complex multistep, multifactorial process 1,2. Experimental carcinogenesis proceeds through at least three distinct stages, in which the first two phases can be used to verify whether the agent of interest acts as an initiator or a promoter. 'Initiation' is the first step and is considered to involve a genotoxic event in which the carcinogen interacts with target cells to affect DNA. This initiating mutation may give the initiated cell the growth advantage needed in the second stage of promotion. In contrast to its neighbors, the initiated cell can escape the cellular regulatory mechanisms. There are four types of initiators: 1) chemical carcinogens (e.g. MNNG, DMBA), 2) physical carcinogens (e.g. X-irradiation, ultraviolet B light), 3) viral agents (e.g. v-Ras) and 4) proto-oncogene activation/tumor suppressor gene loss. 'Promotion' is the second step and is associated with a number of subcellular events that are generally nongenotoxic and is responsible for the conversion or clonal expansion of initiated cells to a cancer. Tumor promoters could either be external or internal stimuli. Only initiated cells are stimulated to grow 3. However, tumor initiation and promotion together produce relatively benign growths. It is the third step, malignant conversion or progression, in which these growths become malignant. This process is generally slow and occurs over a long period of time. 'Malignant conversion', like initiation, requires genetic alteration. Here cellular growth is further deregulated thus proceeds uncontrolled. Progression is probably the most complex of the three stages, because both acquired genetic and phenotypic changes occur, and the cellular expansion is rapid. As the tumor progresses, sensitivity to dietary compounds, inhibitors of growth and enhancers of differentiation gradually disappear until the tumor becomes progressively more autonomous and controllable only by more drastic intervention 4.

Several procedures for evaluating the cell transforming potential of chemicals have been described 5,6,7. In vitro cellular transformation detection assays are semi-quantitative and measure the morphological transformation of cell colonies induced by chemicals 8,9,10. This transformation is associated with certain phenotypic changes such as loss of contact inhibition (cells can grow over one another) and anchorage independence (cells form colonies in soft agar) 11,12. Anchorage independence can be described in the light of primary fibroblasts and many fibroblastic cell lines (e.g. BALB/c3T3, NIH-3T3, etc.) that must attach to a solid surface before they can divide. They fail to grow when suspended in a viscous fluid or gel (e.g. agar or agarose), however when these cell lines are transformed, they are able to grow in a viscous fluid or gel and become anchorage-independent. The process by which these phenotypic changes occur, is assumed to be closely related to the process of in vivo carcinogenesis. Cells derived from transformed colonies after passaging in culture are capable of tumor formation when inoculated in a suitable host animal 8,13.

In general there is reasonably good correlation between in vitro transformation and in vivo carcinogenesis, although the correlation varies depending on the system being studied. These systems are believed to be reasonably good predictors of in vivo activity, and positive results are viewed as potential indications of in vivo carcinogenesis.

CHEMICONâ's Cell Transformation Detection Assay (Catalog number ECM570) is a valuable tool for the morphological and quantitative detection of in vitro malignant transformation of cells induced by certain test compounds. This assay also allows screening of substances that inhibit malignant cell transformation.

Vertebrates

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Cell Based Assays

• Agarose powder (Part No. 90316). One bottle containing 2g of noble agar powder.
• Cell Stain Solution (Part No. 90317). Four vials containing 5mg each of cell stain powder.
• Cell Quantification Solution (Part No. 90318). One vial containing 2 mL of cell quantification solution.

When stored at 2-8º C, the agar and cell stain powder are stable up to the expiration date. Cell stain quantification solution should be stored at -20º C up to expiration date. Discard any remaining reagents after the expiration date.

kit

1. Positive control [e.g. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)]

2. Tumorigenic promoter [e.g. 12-O-Tetradecanoylphorbol 13-acetate (TPA)]

3. Tissue culture dishes and pipettes

4. Cell culture media and supplements

5. 37°C cell culture incubator

6. Microwave or heating block

7. Microscope

8. Spectrophotometer