.

A test system for simultaneous registration of somatic mosaicism for both tumorous and non-tumorous markers. 

Sidorov, R.A.¹, E.G. Ugnivenko², E.M. Khovanova², and G.A. Belitsky¹.  ¹ Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Moscow, Russia;  ² Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia

       Tests for somatic mosaicism in flies heterozygous for recessive markers are widely used to determine mutagenic and prospective carcinogenic activity of different substances. The tests that use as somatic clone markers the genes yellow (y) and singed (sn) or multiple wing hairs (mwh) and flare (flr) or different alleles of white (w) have become classic. Despite the fact that these markers are very useful in somatic mutagenesis studies, they allow only an approximate estimation of the mutagen’s blastomogenic activity, as the clones homozygous for those markers do not develop tumors. To observe tumors, a usual marker should be replaced with a recessive mutation in a tumor suppressor gene, such as the gene warts (wts). In homozygous condition this gene produces tumors in imaginal discs (Bryant et al., 1993;  Xu et al., 1995).  These tumors can be easily observed in wts heterozygous imagoes.

       We have designed a new system that allows observation of both tumorous (wts) and non-tumorous clones (y, sn and twin y–sn) in the same fly. It is based on the female  y; wts^X1 / TM6B, Hu Tb ´ male  w sn cross that produces y + / + sn; wts^X1 / + and y + / + sn;  + / TM6B, Hu Tb females as well as y; wts^X1 /+ and y; + / TM6B males. In this system we have tested three polycyclic aromatic hydrocarbons (PAHs) – benzo(a)pyrene, benzo(e)pyrene and pyrene and cis-dichlorodihydroxydiamminoplatinum IV (oxoplatin), a supermutagenic anti-cancer drug.  The PAHs were dissolved in 10% aqueous dimethylsulfoxide (DMSO). Oxoplatin was dissolved in distillated water.

The 1^st instar F₁ larvae from this cross were treated with the substances (see Table 1 for concentrations). After eclosion, y + / + sn; wts^X1 / + females were screened for tumors and non-tumorous clones, whereas y + / + sn; + / TM6B, Hu Tb females were screened for non-tumorous clones only. Males of all genotypes were rejected.

Clone frequency (p) was calculated as (Number of clones / Number of flies) ´ 100%. Significance of differences between the series was calculated in Student’s t-test with Fisher’s correction .

The results are summarized in Table 1.  The essence of the results is the following.

       1. The new system is sensitive to mutagenic carcinogens of different classes (PAHs and platinum complex compounds). The combination of classical non-tumorous and novel tumorous makers allows the system to recognize even those mutagenic and blastomogenic substances that do not induce clones of some particular type, such as benzo(e)pyrene.

       2. In all series tumors have significantly higher frequency than non-tumorous clones of each type (y, sn or y–sn). In all cases except for benzo(e)pyrene, the tumor frequency is significantly higher than even the total frequency of non-tumorous clones of all types. This suggests that wts is generally a better marker than its non-tumorous counterparts. We suppose that the preponderance of tumor frequency is related to the ubiquitous character of the wts marker, as it does not need any differentiated cuticle structures such as macrochaetae for its manifestation. Also a different somatic recombination rate in chromosomes III and I may contribute to this issue.

       3. The gene wts has no dominant mutator effect on somatic mosaicism for other genes (y and sn) as the frequency of non-tumorous clones is comparable in y + / + sn; wts^X1 / + and y + / + sn; + / TM6B, Hu Tb females that have no wts mutation.

Table 1.  Frequency of tumorous and non-tumorous clones induced by mutagenic carcinogens in heterozygotes for tumorous and non-tumorous recessive markers.

Legend: wts­ – warts clone number; y – yellow clone number; sn – singed clone number; y–sn – twin yellow–singed clone number; S – a total number of non-tumorous clones (yellow, singed and twin yellow–singed); pclone type – clone frequency; *** – the clone frequency is significantly higher than the control one, P < 0.001; ** – the same, P < 0.01; * – the same, P < 0.05. (>) – the tumor frequency is significantly higher (P < 0.05) than the frequency of any particular type of non-tumorous clones. (+) – the tumor frequency is significantly higher (P < 0.05) than the total frequency of non-tumorous clones of all the types. (≈) – the total frequency of non-tumorous clones in y + / + sn; wtsX1 / + heterozygotes has no significant differences from the total frequency of non-tumorous clones in  y + / + sn; + / TM6B, Hu Tb heterozygotes.

       Acknowledgments:  This work was supported in part by the International Science and Technology Center grant # 832 and Russian Foundation for Basic Research grants # 01-04-49285 and # 01-04-49285MAC.

       References:  Bryant, P.J., K.L. Watson, R.W. Justice, and D.F. Woods 1993, Development Suppl.: 239 – 249;  Xu, T., W. Wang, S. Zhang, R.A. Stewart, and W. Yu 1995, Development 121: 1053 – 1063.