Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1993-02-18
1995-02-14
Weimar, Elizabeth C.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
435 72, 435 721, 435 74, C12N 506, C12N 1500
Patent
active
053895410
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to new erythroblastoid mouse cell lines.
The properties of lethally irradiated mice, reconstituted over a long period of time with bone marrow cells which express the src gene (after infection with the N-TK-v-src retrovirus) are already known (Keller and Wagner, 1989). Primary recipient mice of this kind develop a severe myeloproliferative disease characterised by a large number of erythroid precursor cells (in addition to increased numbers of precursor cells of other lineages).
Attempts to cultivate such cells in vitro have not hitherto resulted in the establishment of permanent haematopoietic cell lines.
The aim of the present invention was to provide erythroblastoid cell lines having specific properties.
This aim was achieved by transferring spleen cells from primary recipient animals once again into lethally irradiated mice (secondary recipient animals). To do this, nucleus-containing spleen cell preparations (i.e. those free from erythrocytes) were injected intravenously into lethally irradiated CBA mice (Keller and Wagner, 1989). Three week later the secondary recipient animals were killed and cells from spleen and bone marrow were cultivated in the presence of interleukin-3 (IL-3) and human recombinant erythropoietin (EPO). After 10 days, in addition to normally differentiated colonies of erythroid and myeloid origin, large colonies consisting of undifferentiated, blast-like cells developed in these cultures. These colonies could not be found in corresponding bone marrow cultures of control animals. A fairly large number of these colonies were isolated and expanded in the presence of EPO and IL-3. After an initial period of slow growth, some of the cultures obtained were able to be grown into mass cultures and finally established as cell lines. In view of the fact that they originate from individual colonies they can be regarded as clonal.
It was found, surprisingly, that the cell lines thus obtained survive and proliferate exclusively in the presence of erythropoietin. This strict dependency on EPO was found in all the cell lines investigated, the degree of EPO dependency being found to differ. EPO dependency can therefore be regarded as a characteristic feature of these cell lines which express the src oncogene.
The present invention thus relates to new erythroblastoid mouse cell lines which express the src gene and which are strictly erythropoietin-dependent for their survival and proliferation.
According to investigations which included three further cell lines in addition to the cell line designated E 4 and E 31 which were first to be characterised, it was found that the cell line E 31 shows the greatest EPO dependency. The other cell lines are dependent on erythropoietin to varying degrees. All in all, it was established by means of the experiments carried out that the dependency on erythropoietin is a common feature of cell lines which express the src oncogene.
The result that the cell lines E 4 and E 31 respond strongly to erythropoietin, a growth factor which acts specifically on late erythroid precursor cells, led one to assume that the cells of the erythroid lineage can be put down to haematopoietic differentiation or at least represent determined erythroid precursor cells (committed progenitors). In order to demonstrate this, the cells were investigated for two markers specific to erythroid cells, namely haemoglobin and the mamalian-homologous histone protein (presumably H1-0) of the erythroid-specific hen's histone H5.
The investigations into haemoglobin production and the presence of the mammalian homolog of histone H5 clearly showed that the cells of lines E 4 and E 31 belong to the erythroid lineage.
In order to demonstrate that all cells of cell lines E 4 and E 31 are of erythroid descent, attempts were made to induce haemoglobin production by means of chemicals. Whereas other erythroleukaemic mouse cell lines such as Friend cells (Orkin et al., 1975) can be induced by means of 2% DMSO to produce haemoglobin in more than 80% of cells, hen's erythroblast
REFERENCES:
Keller et al. 1989. Genes and Development 3:827-837.
Lewis et al. 1989. Exp. Hematol. 17:102-105.
Amersham Corp., Life Sciences Products Cataloge, 1989/90 Ed. p. 40.
Beug Harmut
Keller Gordon
Wagner Erwin
Boehringer Ingelheim International GmbH
Frankhouser D. E.
Stanton Brian R.
Stempel A. R.
Timbers M-E. M.
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