Table 1.
Carcinogen-specific induction of genetic instability
| Clone name | Description | Parental cells | CIN,* % | PhlP† | MNNG† |
|---|---|---|---|---|---|
| H3 | Parent culture | H3 | 3 | <1 | 1 |
| Control 8 | unselected control subclone | H3 | 1 | 2 | <1 |
| Control 14 | unselected control subclone | H3 | 3 | 4 | 2, 3 |
| Control 17 | unselected control subclone | H3 | 1 | 3 | <1 |
| P1 | Cloned after treatment with PhlP | H3 | 16 | 73, 63 | 60 |
| 2P1 | Cloned after treatment with PhlP | H3 | 42 | 44, 39, 47 | <1 |
| 2P2 | Cloned after treatment with PhlP | H3 | 33 | 49, 21 | 8 |
| 2P4 | Cloned after treatment with PhlP | H3 | 30 | 10 | 5, 6 |
| 2P7 | Cloned after treatment with PhlP | H3 | 12 | 21, 12 | 5 |
| P2 | Cloned after treatment with PhlP | H3 | 20 | 10 | 3 |
| P3 | Cloned after treatment with PhlP | H3 | 22 | 50 | <1, <1 |
| 2P9 | Cloned after treatment with PhlP | H3 | 25 | 45, 33 | 5, <1 |
| A6 | Cloned after treatment with MNNG | H3 | 1 | 31 | 58 |
| 5N | Cloned after treatment with MNNG | H3 | 5 | <1 | 37 |
| N68 | Cloned after treatment with MNNG | H3 | 3 | 7 | 27 |
| N70 | Cloned after treatment with MNNG | H3 | 2 | <1 | 62, 80 |
| N74 | Cloned after treatment with MNNG | H3 | 5 | <1 | 42, 65 |
| A2 | Cloned after treatment with MNNG | H3 | 10 | <1 | 70, 68 |
| A5 | Cloned after treatment with MNNG | H3 | 5 | 18 | 63 |
| GFP-DLD1, uninduced | no exogenous expression | DLD1-TET | 2 | <1 | ND |
| GFP-DLD1, induced | express GFP only | DLD1-TET | 1 | <1 | ND |
| BUB-DLD1, uninduced‡ | no exogenous expression | DLD1-TET | 4 | <1 | 51 |
| BUB-DLD1, induced‡ | express GFP + mutant hBUB1 | DLD1-TET | 34 | 17, 38 | 47 |
Representative clones derived from treatment with the indicated agents were studied for chromosomal instability (CIN) and resistance to carcinogens. H3 cells are an HCT116 derivative carrying an extra copy of chromosome 3 that contains the wild-type hMLH1 gene (7). DLD1-TET cells are a derivative of DLD1 cells that constitutively express tTA, a modified tet-based transcriptional activator (23).
Chromosomal instability was measured by FISH analysis, with the numbers indicating the fraction of cells whose chromosome 12 copy number was different from the modal number of the clone, analyzed 25 generations after single cell dilution. The modal number was 2 in the diploid clones and 4 in tetraploid clones (see Materials and Methods). Boldface numbers indicate values of CIN, % > 10.
The number of colonies arising after treatment of the indicated clones with PhlP or MNNG relative to the number of colonies arising in control flasks in the absence of treatment. ND, not determined.
GFP-DLD1 and BUB-DLD1 cells were derived from DLD1 cells engineered to inducibly express genes under the control of a tet operon. GFP-DLD1 cells inducibly express GFP, while BUB-DLD1 cells inducibly express a dominant mutant hBUB1 gene in addition to GFP. As DLD1 cells are MMR deficient, they are resistant to MNNG, irrespective of induction.