C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype

Abstract

We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.

Keywords: C. elegans; MET; MT13032; PS1258; PS2728; RB2088 and nicotine; SD551; vab-1.

Figure 1.

Effect of Nicotine on survival ofC. elegansSurvival plots of C. elegans when exposed to different concentrations of nicotine are shown. The experiment was repeated twice and the results were comparable. The data was subjected to Kaplan Meier survival analysis using Graphpad Prism Software 4.02V.

Figure 2.

Effect of Nicotine on egg-laying capacity and fertility ofC. elegansa. Egg-laying responses to chronic exposure of nicotine were assayed on NGM agar. Each condition had 24 worms and the assay was repeated 4 times. Rate at which eggs were laid was calculated as eggs/worm and percent relative egg-laying compared to untreated N2 worms is shown. b. To quantify the number of progeny produced by hermaphrodites, synchronized L4 worms were collected and at least 4 worms were allowed to lay eggs on individual plates. Animals were examined until no progeny were produced within a 24-h period and percent relative fertility compared to untreated N2 worms is shown.

Figure 3.

Phenotypic changes inC. elegansafter chronic treatment with nicotine. Phenotypes of SD551 mutant. 10X and 20X magnification images of SD551 a. control showing normal vulva phenotype and b. with nicotine showing multiple vulva phenotype. Phenotypes of PS2728 and MT13032 (Sli-1) mutants. 10X and 20X magnification images of PS2728 c. control showing normal body size and d. with nicotine showing shrinkage in the body size (MT13032 not shown).

Figure 4.

(A). Effect of chronic exposure of nicotine on the locomotion velocity of differentC. elegansstrains. Synchronized worms were grown on plates with and without nicotine. 20-30 L4 worms were transferred to bacteria free plates with nicotine and incubated for 20 min before recording tracks. Worms were tracked for 600 seconds. The corresponding normalized histograms of N2, SD551, vab-1, RB2088, MT13032, PS1258 and PS2728 speeds with (a, c, e, g, i, k, m) and without nicotine (b, d, f, h, j, l, n) are respectively shown. Average velocity (mm/s) of each mutant is shown in Table 3. (B). Effect of chronic exposure of nicotine on worm path. Worms (N2 wt, and vab-1) were tracked for 10 minutes. The tracks of 20 worms with and without nicotine are shown.

Figure 5.

Effect of Nicotine on Gene Expression analysis of N2,vab-1and SD551 mutants ofC. elegans. Panels a-c: Principle Components Analysis plots of the first 3 PCAs. The letter designates the genotype of the strain (v = vab-1, s = SD551, n = N2) and the color designates nicotine exposure (blue = 500 x nicotine, red – 0 x nicotine). Panels d-f: -log10 p-value (fisher’s exact test) for enrichment of pathways and process in differentially expressed genes. Panel d shows nicotine alone, Panel e show the interaction between nicotine and vab-1, and Panel f shows the interaction between nicotine and SD551.

Figure 6.

Effect of Nicotine on heatmap analysis of N2,vab-1and SD551 mutants ofC. elegans.Panel a: Heatmap of β effect sizes from linear models for differentially expressed genes in the “positive regulation of growth rate." Panels b-d: Beta effect sizes for 3 example genes in “positive regulation of growth rate." (Red = N2, Blue = vab-1, Green = vab-1+500 Nicotine, Purple = SD551, Orange = SD551+Nicotine, Yellow = 500 Nicotine). Panels e-f: Heatmap of gene expression levels for differentially expressed serine-threonine kinases.