Previous experiments have demonstrated that antigens present in Xenopus laevis embryos are shared with human malignant neoplasms. In fact it is known that antisera raised in rabbit against pellet and supernatant fraction of Xenopus laevis embryos react to different antigens present in human tumors. The aim of the present study was to evaluate whether these antisera react also to different antigens contained in zebrafish embryos at different stages of cell differentiation. This was done with the agar-gel immunodiffusion method performed according to an already described protocol.
The results that we obtained show that both antisera raised in rabbit against pellet and supernatant fractions of Xenopus laevis embryos react to antigens of zebrafish at different stages of cell differentiation.
It has already been described that antibodies produced by immunizing rabbit to Xenopus laevis embryo fractions react with a variety of human malignant neoplasms. In fact antisera raised in rabbit against pellet and supernatant fractions of Xenopus laevis embryos react to different antigens contained in 50 of 55 different tumors. These reactions were demonstrated histologically.1
The antigens in the supernatant fraction are probably different from those in the pellet fraction. Antisera raised against the pellet fraction react with carbohydrate antigens.2 The nature of the antigens in the supernatant is not known yet. These previous results suggested that Xenopus laevis ambryos may contain an “oncodevelopmental” carbohydrate re-expressed in human tumors and that Xenopus may be an useful source for tumor associated antigens of human malignant tumors.
It may be that the antigens of Xenopus laevis embryos are preserved during phylogeny and that they are shared with other different embryos. The aim of the present study is to evaluate if the antisera raised in rabbit to the pellet and supernatant fractions of Xenopus laevis embryos react with antigens of zebrafish embryos at different stages of cell differentiation.
fig. 1 |
fig. 2 |
fig. 3 |
Figure 1
Zebrafish: middle - blastula - gastrula
Antitsera R 750-I (supernatant) A R 755-I (pellet) B
Figure 2
Zebrafish: 5 somites
Antisera R 750-I (supernatant) A R 755-I (pellet) B
Figure3
Zebrafish: 20 somites
Antisera R 750-I (supernatant) A R 755-I (pellet) B |
Materials and Methods
The embryos of zebrafish at the stages of middle-blastula-gastrula, 5 somites, 20 somites were washed in distilled water and placed in a solution of pure glycerol and 30% of ethanol at a 4:1 ratio. The embryos were sonicated with 2 cycles of 10 seconds each and further treated with a turboemulsifier. 35 microliters of these solutions were used in an agar-gel immunodiffusion test. The methods of preparation of antisera raised in rabbit to pellet and supernatant fractions of Xenopus laevis embryos have already been described.1,2 A solution of 1 gram of agar-gel diluted in 40 ml. of distilled water and in 10 ml. of TBE (Tris Boric acid EDTA) was put in a plastic support. The lyophilized antisera were resuspended in 500 microliters of distilled water and 35 microliters of this solution were distributed in different wells of agar gel. 35 microliters of extracts of zebrafih at different stages of cell differentiation were put in different wells, in front of the antisera at a distance of 1 cm. The agar-gel with antisera and embryonic extracts were incubated for 24 hours at 22 degrees centigrade. The agar gel was stained with Coomassie Blue for 15 minutes, then washed with distilled water and finally destained in a solution composed by 10% acetic, 45% ethanol, 45,% distilled water.
Results
The results that we obtained show that both antisera raised in rabbit to pellet and supernatant fraction of Xenopus laevis embryos react to antigens of zebrafish at the three stage of cell differentiation. In fact the Fig. 1, Fig.2 and Fig.3 show that the antigens of zebrafish embryos at the stages of 50% of epiboly, 5 somites, 20 somites react with antisera R750-1 raised in rabbit to supernatant (part A of the figure) and with antisera R755-1 raised in rabbit to pellet (part B).
Conclusions
These results suggest that Xenopus laevis embryos share antigens with zebrafish embryos. These antigens are conserved during phylogeny. They are expressed in the zebrafish embryo since the beginning of cell differentiation and are present until last organogenesis. These antigens are more expressed at the end than at the beginning of organogenesis, as the results illustrated in Fig. 3 show in comparison with the results of the Fig. 1 and 2. In any case, these antigens shared with different species of embryos are re-expressed in different human tumors as already described. Tumor cells re-express several others “oncodevelopmental” antigens in addition to those described from us. For this reason, tumor cells can be considered as mutated embryonic cells, in which “gene configurations” are similar to those present in embryo during the steps of multiplication comprised between two stages of cell differentiation. This hypothesis has been already put forward in previous reports.3,4,5,6,7,8 On the other hand, tumor cell genome is normally affected by a dramatically high number of altered genes, most of which playing an important role in normal embryo development. In fact, during tumorigenesis some embryonic genes are activated or mutated, leading the cell to an uncontrolled multiplication program.
Many substances present in embryo during organogenesis are able to reduce tumor growth in vitro or in vivo, because they regulate some important genes that control cell differentiation and multiplication.5,7 In fact, our previous works demonstrated that substances present in embryo during cell differentiation are able to reduce tumor growth in vivo4 and to activate the tumor suppressor p53 in different tumor cell lines in vitro.5 Other works led to the same hypothesis: in fact the transplantation of teratocarcinoma cells into the mouse blastocyst origins normal chimeric mice, since the teratocarcinoma cells are led to differentiate in various kinds of tissues9 Otherwise, the transplantation of embryonic stem cells in mice origins teratocarcinomas.10 Our results suggest that Xenopus laevis and zebrafish embryos may be an useful source for tumor associated antigens of human malignant tumors.
P. M. Biava,
Ospedale Civile SSG Milano,
Italia
A. Monguzzi,
Ospedale Civile SSG Milano,
Italia
D. Bonsignorio,
Ospedale Civile SSG Milano,
Italia
A. Frosi,
Ospedale Civile SSG Milano,
Italia
S. Sell,
Albany Medical College,
Albany, NY,USA;
J. V. Klavins,
Albert Einstein College Of Medicine,
New York, NY, US;
