DE19612463A1 - New protein with differentiation-inducing activity for erythropoietic cells - Google Patents

Abstract

A new protein is disclosed having at least a differentiation-inducing activity, in particular for Friend's erythroleukemia cell lines.

Description

The invention relates to a new protein with differentiation inducing activity derived from mammalian cells, in particular the culture supernatant of mammalian cells can be isolated. The Protein is especially from murine and human cells isolatable.

In the culture supernatant of a murine myelomonocytic leukemic Cell line (WEHI-3) was found to be an erythroid activity Mouse cells (Friend erythroleukemia cell lines) for differences ornament induced with hemoglobin formation. The molecular weight this activity is between about 10 and 60 kDa, so that ver separated large protein species or aggregates of activity are increasing. The expression of the activity depends on a previously uncharacterized serum factor that is found in different concentrations in different batches of commercial fetal calf serum. One regarding the  Effect identical activity was also irradiated in the supernatant ter human bone marrow stromal cells (cell line L88 / 5) found. The activity received the preliminary name EDA (Erythroid Differentiation Activity), although other than erythropoiesis inducing functions from the expression pattern of the belonging gene can be derived (see below). On the human leukemia cell line K562 also failed erythro Poese-inducing effect of EDA can be demonstrated (Spe cross-cut effect of EDA).

A small RNA species of the Gens isolated after rewriting in cDNA (DY-8), which according to Trans fection in Cos-1 cells led to a culture supernatant which Had EDA activity. With this 720 bp cDNA as a probe a larger 1,350 bp piece of cDNA was isolated (HA-15/2), that with temporary expression in Cos-1 cells and use of the Cos-1 supernatant also had a weak EDA effect pointed. The gene to be patented is in the cells examined the mouse in different RNA species (probably Spleißva 800, 1,200, 1,350, 1,750 and 2,200 bp expri lubricated. An eda mRNA expression in sometimes very low order catch was found in all examined tissues (liver, Kidney, brain, intestine, placenta). The strongest expression takes place in the thymus, followed by fetal liver, adult spleen and Bone marrow. Many mouse haemopoietic cell lines, especially leukemic transformed like DA-3, WEHI-3, NFS-60 or NFS-61, but also not transformed leukemically like NIH-3T3 or TS1-C3, show some very strong expression at the RNA level by eda.

Examining the importance of eda in normal spleen cells the mouse showed that a mitogenic or T cell receptor-specific stimulation of the cells or stimulation of the protein kinase C does not lead to stable expression of the gene. On the other hand  Expression occurs when an allogeneic spleen cell reaction with unirradiated, not pretreated spleen len the mouse strains CBA and C57Bl6 is carried out. The one here deductible meaning of participation in the allogeneic Response of the spleen cells was determined by a semi-allogeneic trans plantation model of the mouse (C57Bl6 spleen cells in lethal be beamed CBA × C57Bl6 mice injected) underlined. In the mil zen of the experimental animals it occurred in the course of the acute "graft ver sus host disease compared to that of the tissue contract transplanted controls to an approximately 7-fold increase the eda expression.

The 2.2 kbp band of the eda mRNA shows the most constant expression pattern in all experiments. Their structural structure, analyzed on NIH-3T3 cells, is shown in Fig. 18. The repeat structures are an important characteristic of the eda cDNA. The associated sequence, a consensus sequence from clones of the WEHI-3 and the NIH-3T3 cell lines, is shown in Fig. 17, which corresponds to SEQ ID NO: 1. All mRNA species, i.e. the 300, 1,200, 1,350, 1,750 and 2,200 bp bands, have an identical 3'-end, referred to as "tail" in Fig. 18, and an only short band-specific 5'-part. The erythropoiesis-differentiation-inducing effect was found with the clone DY-8, which approx. 640 bp of the 3′-region of eda and also approx. 75 bp of its own 5′-end ( Fig. 19 corresponds to SEQ ID NO: 2) contains. The differentiation-inducing function is thus at least partially bound to the 3'-part of the cDNA.

Comparisons of the band sizes of the different eda mRNA species in different mouse strains such as Balb / c, C3H, CBA, C57Bl6, Swiss, AKR or NFS as well as partial sequences of the eda cDNA from different mouse strains leave a mouse strain dependent Recognize variability. So this gene probably comes meaning for histocompatibility. The strenght  Expression of eda in some mouse tumor cell lines leaves one Expect importance for tumor growth.

Although eda is expressed in many cell lines, one can find in the culture supernatants from very few cell lines EDA-Akti vity. The presumptively different protein species are for this usually follow anchored to the cells. A function the cell surface in the context of specific cell-to-cell inter actions (differentiation induction, allogeneic reaction) to assume at the current state of the investigations.

With the help of the murine eda Probe HA-15/2, it was possible to achieve this human gene at both the DNA level by Southern Blot analysis under stringent conditions, as well as on RNA level ne by Northern blot analysis. Here was especially the Jurkat cell line is positive. In addition, one clear band of approx. 1,100 bp in a case of human chonic T-helper cell type T cell leukemia found, that in no other of 8 samples of human cell lines and primary human bone marrow material was recognizable. Even if the aetiological significance of this finding of a white This finding opens up the possibility of further clarification a therapeutic and diagnostic use of tools conditions for certain forms of human malignancies that are caused by Developable due to the data contained in this release will be.

It was an object of the present invention, a new one Protein with differentiation-inducing activity, esp especially for erythropoietic cells.

This object was achieved by the provision of the protein with differentiation-inducing activity solved the following properties:

• a) isolable from murine myelomonocytic leukemic Cell lines;
• b) isolatable from irradiated human bone marrow stream macelles;
• c) induces differentiation in friend erythroleukemia cell lines with hemoglobin formation;
• d) with a molecular weight in the range of about 10-60 kDa;
• e) inducible by an occurrence in the fetal calf serum emitting serum factor;
• f) with an expression of the associated mRNA in primary Cells from thymus, fetal liver, adult spleen or Bone marrow;
• g) with a stable expression of the associated mRNA in vitro if an allogeneic spleen cell reaction with unbe radiated, not pretreated spleen cells from the mouse strains CBA and C57Bl6 is performed;
• h) with characteristic repeat structures in the for the Protein encoding cDNA;
• i) with AT-rich sections in the for the protein coding cDNA;
• k) with associated mRNA species of different sizes, the from the same 3'-areas, but different 5'-Be rich exist.

The protein provided according to the invention is from mammal cells, preferably from the supernatant of mammalian cell cultures, isolatable. In preferred embodiments of the invention isolated from murine or human cells. Preferred celli are: the murine myelomonocytic leukemic cell line WEHI-3, ATCC TIB68 and the irradiated human bone marrow stroma cell line L88 / 5, DSM ACC 2056. Friend erythroleukemia cell lines F4N or B8 / 3 serve to detect the protein.  

The protein provided according to the invention at least has a partial amino acid sequence derived from one with the cDNA of SEQ ID NO: 1 or NO: 2 hybridizing DNA is encoded. The hybri doping is preferably carried out under stringent conditions.

Stringent conditions in the sense of the present invention defined as such conditions that are selective and detectable specific binding of the nucleic acid to the for the gene or transcripts encoding the protein of the invention of the gene coding for the protein according to the invention lichen. Such a hybridization under stringent conditions preferably means that after hybridization 65 ° C in an aqueous solution or at 42 ° C in 50% formamide and then washing the filter at 60 ° C in an aqueous Solution with a salt concentration of 15 mM NaCl and one SDS concentration of 0.1% still binds the probe to the for the gene coding for the protein according to the invention or the here of derived RNA is detectable. When using brevity However, if nucleic acids are used as probes, less drastic hybridization and / or washing conditions to use.

Parts, analogs and Derivatives of the protein according to the invention and fusion proteins includes. The protein according to the invention is preferably in essentially purified and native form or in essentially Chen recombinant form.

The protein according to the invention has a differentiation ind ornamental activity for erythropoietic cells. It is however, based on the available investigation results thereof assume that this differentiation-inducing activity not only for erythropoietic cells, but also for others Cells applies.  

It has been found according to the invention that the provided Protein has an erythropoiesis-inducing effect on the human leukemia cell line K 562 (ATCC No. CRL243).

The present invention comprises DNA fragments according to SEQ ID NO: 1 or NO: 2, parts, derivatives and analogues thereof and DNA frag elements, parts, analogs and derivatives thereof which are associated with the cDNA according to SEQ ID NO: 1 or NO: 2, preferably under stringent Conditions, hybridize.

The present invention also relates to DNA fragments that encode at least a portion of a polypeptide which the activity of the human or murine protein with Differentiation-inducing activity for erythropoietic Has cells according to the present invention.

The present invention further relates to recombinant Vectors containing a DNA sequence that a gene or corresponds to a DNA fragment that is suitable for a protein with diffe induction-inducing activity for erythropoietic cells encoded according to the invention. In the vectors according to the invention can be conventional, known from the prior art Act vectors, for example bacterial plasmids, Bacteriophages or viral vectors.

The invention further includes host cells with a vector provided according to the invention are transformed. At the host cells can be prokaryotic or eukaryotic cells, for example E.coli cells or yeast cells.

The DNA fragments according to the invention according to SEQ ID NO: 1 or NO: 2, parts, derivatives or analogues thereof, are invented obtained according to, for example, that a human or murine cDNA clone library using a DNA fragment  a DNA that is for a murine or human protein differentiation-inducing activity coded, screened becomes.

The present invention further relates to monoclonal or polyclonal antibodies against the protein of the invention with differentiation-inducing activity for erythropoeti cells, a part, a derivative or an analog of this are directed.

According to the invention, a therapeutic, diagnosti means or experimentally usable means that as active substance at least one nucleic acid in one contains the same amount with a gene or a part thereof hybridizes, and with the protein according to the invention Differentiation-inducing activity for erythropoietic Cells coded.

According to the invention, a therapeutic, diagnostic or experimentally usable means that is characterized in that it is at least one as an active substance Contains nucleic acid, which (a) for a protein with differen ornament-inducing activity for erythropoietic cells coding nucleotide sequence, (b) a part thereof, (c) a with a nucleic acid from (a) and / or (b) under stringent Conditions hybridizing nucleotide sequence or (d) one to a nucleotide sequence from (a), (b) and / or (c) complementary Nucleotide sequence comprises. The nucleic acid of the agent is one possibly modified DNA or RNA.

The therapeutic agent provided according to the invention ent keeps the protein with differentiation-inducing activity for erythropoietic cells of the present invention Analogue, derivative or parts thereof together with usual  Carriers and / or auxiliaries in an effective amount.

The therapeutic, diagnostic, provided according to the invention means or experimentally usable means is, for example, as molecular probe in diagnostics or as an antisense nucleus acid can be used to inhibit gene expression. By using Antibodies against this agent can be used therapeutically, diagnostic or experimental influence on the differenzie tion-inducing effect.

The invention further relates to a method for transforming tion of a prokaryotic or eukaryotic cell using a DNA, which is for the protein provided according to the invention encoded with erythropoiesis inducing activity, as well as parts, Derivatives or analogues of this DNA.

The invention further relates to a fusion protein with a Amino acid sequence consisting in whole or in part the amino acid sequence of human or murine protein with Differentiation-inducing activity for erythropoietic Cells according to the invention and part of a prokaryonti human or eukaryotic protein.

The invention further relates to a synthetic protein Differentiation-inducing activity for erythropoietic Cells according to the invention with an amino acid sequence which is from a DNA sequence that is encoded with the DNA sequence SEQ ID NO: 1 or NO: 2 at least under stringent conditions hybridizes.

The protein provided according to the invention is suitable before moves to the treatment of diseases with disorders in the dif erythropoietic cell-inducing activity.  

The invention based on the present Abbil with reference to preferred embodiments described in more detail. However, the invention is not based on the described preferred embodiments limited.

The pictures show:

Fig. 1 Erythroid induction of differentiation in murine F4N erythroleukemia cells through 4 different WEHI-3 conditioned media.

Fig. 2 Erythroid induction of differentiation in murine B8 / 3 erythroleukemia cells by culture supernatants from the human stromal cell line L88 / 5, measured on days 3 and 4.

Fig. 3 Erythroid induction of differentiation in human K562 CML cells by 4 different WEHI-3 conditioned media. An effect can be seen in particular with the WCM (C).

Fig. 4 Effect of WEHI-3 conditioned medium on cell number and α-globin synthesis of B8 / 3 mouse Erythroleu kemia cells.

Fig. 5 Effect of WEHI-3 conditioned medium on the adherence of WEHI-3 cells to the plastic base of culture vessels with a culture duration of 72 hours.

Fig. 6 Gel-chromatographic fractionation of WEHI-3-conditioned medium with Sephacryl S 300® and biological testing for EDA activity with B8 / 3 mouse Ery throleukemia cells in a culture period of 4 days. The induction of α-globin mRNA was measured by Northern blot analysis.

Fig. 7 Dependence of EDA production on WEHI-3 cell density when harvesting from the primary, FCS-containing culture. The activity was determined by culturing B8 / 3 mouse erythroleukemia cells with the WEHI-3 supernatants for 3 days and counting the percentage of benzidine-positive cells.

Fig. 8 Fractionation of the supernatant from experiment W3 / 2 (see Fig. 7) and testing of the fractions by 3-day cultivation with B8 / 3 mouse erythroleukemia cells and counting of the percentage of benzidine-positive cells.

Fig. 9 Steps of expression cloning of a murine eda sequence by gradually reducing the number of clones from the expression library included in the biological test.

Fig. 10 Expression of the incomplete eda clones HA-15/2 and HA-12/1 in Cos cells and testing of the Cos supernatants in B8 / 3 mouse erythroleukemia cells in culture batches over 3 and 4 days. The percentage of benzidine-positive cells was evaluated. On day 4, the difference between the control and the result with the clone HA-15/2 is just below the 5% significance limit.

Fig. 11 Southern blot analysis of various EcoRI-cut mouse DNA's with the murine eda sample from HA-15/2. The DNA from the human K562 cell line (4th lane from left) was also analyzed, which shows a weak positivity by bands at approx. 7.5 and 6.5 kbp with a highly stringent wash.

Fig. 12 eda expression in the RNA of the murine cell lines NIH-3T3 and M2-10B4. The hybridization in the Northern blot was carried out with various samples as described in the text.

Fig. 13 Northern blot analysis of the RNA of various human cell lines with the murine eda sample from HA-15/2. For the cell lines H33, Reh and K562 15 µg total RNA were used, for the Jurkat sample approx. 5 µg poly (A) + RNA.

Fig. 14 eda expression in murine spleen cells (strain C3H) after stimulation with anti-T cell receptor antibody or concanavalin A. The densitometric results of the evaluation of Northern blots after hybridization with the murine eda sample from HA-15/2 are shown separately for the large eda bands of 2,200 and 7,000 bp, as well as for the smaller, degradation products significant bands of 600 and 400 bp.

Fig. 15 eda expression in murine spleen cells of the mouse strains CBA and CBL when performing a 3-day mixed spleen cell cultivation. Controls are the eda expression in murine placenta and murine fetal liver (d 15). Northern blot analysis with 5-15 µg total RNA and using the eda clone HA-15/2 as a probe. The loading of the individual webs can be read from the subsequent hybridization of the RNA samples of the same filter with a probe for 28 S-RNA.

Fig. 16 Expression of the 2.2 kbp eda mRNA species in semi-allogeneic (CBL cells in (CBA × CBL) F 1 hybrids) or isologous transplantation of CBA × CBL spleen cells into the same recipient types. 5 × 10⁷ spleen cells were transplanted per animal. The eda expression of CBL cells on day 0 was set 100%. Northern blot analysis of 15 µg total RNA per lane with the murine eda sample from HA-15/2.

Fig. 17 Consensus partial sequence of the 2,200 bp eda cDNA. This sequence does not have a continuous open reading frame, so that the absence of individual sections within the sequence is possible.

Fig. 18 Structure of the 2.2 kb eda cDNA. Abbreviations: R 1-1 to R 1-3 = 81 bp repeats 1-3; R 2-1 and R 2-2 = approx. 180 bp repeats 1 and 2; Tail = 3′-end of the gene, identical for all mRNA species; Unknown = approx. 500 bp previously unknown sequence; Begin? = 81 bp section from the 5 'region, the exact location of which has yet to be determined within the initial sequence of the 2.2 kb eda cDNA.

Fig. 19 Sequence of the 715 bp clone DY-8. The first 73 bp (italics) determine the 5′-end specific for this RNA species (this may not be complete yet). The coding sequence is shown in bold.

The attached tables show:

Table 1: The percentage reductions in EDA assets act.

Table 2: Cytokines without differentiation-inducing stocks vity on mouse erythroleukemia cells.  

Table 3: Repeat structures in the presumptive consensus Partial sequence of the 2,200 bp eda DNA.

material and methods Cell lines

Studies were carried out with the following cell lines:
The murine myelo-monocytic leukemia cell line WEHI-3 (Warner et al., 1980, ATCC No. TIB 68), the murine embryonic fibroblast line NIH-3T3 (ATCC No. CRL 1658), the human line which resulted from chronic myeloid leukemia K562 (ATCC No. CRL 243), the human ALL cell line Reh (Rosenfeld et. Al., 1975, ATCC No. CRL 8286), the line H33HJ-JA1 (ATCC No. CRL 8163) derived from the Jurkat cell line, and the monkey kidney cell lines Cos-1 (ATCC CRL 1650) and Cos-7 (ATCC CRL 1651) were purchased from ATCC. The murine cell lines NFS-60 and NFS-61 (Holmes et al., 1985), DA-3 (Ihle et al., 1984) and FDCP-1 (Dexter et al., 1980) are from J. Ihle, Dept. of Biochemistry's, St. Jude’s Hospital, Memphis, Ten., USA The murine mye loische line 32DCl23 (Greenberger et al., 1983) as well as the murine T helper cell line TS1-C3 (Uyttenhove et al., 1988) and the murine thymoma line EL-4 (Farrar et al., 1983) was provided by L. Huelner from the GSF Institute for Experimental Hematology. The mast cell line L138.8A also came from him (Huelner et al., 1989). Murine Friend Erythroleukemia cell lines F4N and B8 / 3 (Ostertag et al., 1974) were provided by W. Ostertag, Department of Virology, Heinrich Pette Institute, Hamburg. The human T-lymphoma cell line Jurkat was developed by S. Thierfelder, GSF-Inst. f. Immunologie, München, and the murine bone marrow stromal cell line M2-10B4 (Le moine et al., 1988) is from C. Eaves, Terry Fox Institute, Vancouver, Canada. The human bone marrow stromal cell line L88 / 5 was established in our institute from the bone marrow of a healthy donor by transfection with a replication-deficient SV-40 virus construct (Thalmeier et al., 1994).

Mice

All mice were from GSF's own pathogen-free breeding pulled. The following strains were used: C3H, Balb / c, CBl (C57Bl6), CBA and AKR.

Media for cell culture

The media were from Life Technologies, D-76339 Eggenstein. All cells with the exception of the Cos-1 and Cos-7 cells were cultivated in RPMI-1640 with 2 mM 1-glutamine and 100 U / ml penicillin and 100 μg / ml streptomycin (Life Technologies). This medium also contained 10% fetal calf serum, which we obtained from various manufacturers and specially tested for the particularly desired properties (e.g. support for differentiation induction or optimal proliferation). In order to obtain WEHI-3 conditioned medium, a serum-poor medium from RPMI-1640 medium was produced analogously to the information provided by Guilbert and Iscove (1976). This contained 0.1% bovine serum albumin, 2 mM 1-glutamine, 20 U penicillin / ml, 20 µg / ml streptomycin, 32 mg / ml iron-saturated human transferrin, 10 ^-5 M 1-α-dipalmitoyl lecithin, 2 × 10 ^-5 M oleic acid and 2 × 10 ^-5 M cholesterol. For the cultivation of Cos cells Dulbecco's medium with 4.5 g / l D-glucose, 10% FCS, 2 mM 1-glutamine and 100 U / ml penicillin and 100 µg / ml streptomycin was used.

Bacterial strains

The strains DH5 (Life Technologies, D-76339 Eggenstein) and Su  re® (Stratagene, D-69044 Heidelberg) were used.

Bacterial media

The bacteria were routinely grown in LB medium (Sambrook et al., 1989). For transformation (Hanahan, 1983) also SOC medium according to the information from Sambrook et al. (1989) used.

Vectors

As a prokaryotic-eukaryotic shuttle vector and express sionsplasmid was a 3,753 bp vector primer after the Principle of Okayama and Berg (1982) from USB, Cleveland, Ohio, U.S.A. used. This plasmid pXRSP + (Pruitt, 1988) contains an SV40 Origo. The gene expression is in eukaryonti cells controlled by the early SV40 promoter. Also ent it holds an SV40 polyadenylation site.

In addition, a gene library was created in lambda phages uses the vector Lamda-Zap II® (from Stratage ne, La Jolla, CA., U.S.A.). This lambda phage allows for Use of a suitable bacterial strain, e.g. B. Sure®, the Blue-white selection and contains a plasmid (pBluescript® SK-), by the helper phage R408 together with the insert from the λ phages can be cut out.

Methods

For cell or organ removal from mice, the animals were in Etheric anesthesia killed. The organs came for the RNA extraction immediately in liquid nitrogen and were from this by Zer reduction in the frozen state in a pre-cooled mortar worked up further with the addition of guanidine rhodanide buffer. Bone marrow cells were taken with a 12-needle using RPMI medium injected without serum from the diaphyses of the femur and tibia and passed through a sieve. Spleen cells were removed by rough cutting  the organs with scissors and by rubbing Sieve using a syringe plunger with the addition of RPMI-Me dium obtained without the addition of serum.

The total RNA from cells was determined by acid phenolization the method of Chomczynski and Sacchi (1987). Out of it if necessary, the mRNA was hybridized to oligo (dT), which was bound to magnetic particles via streptavidin-biotin (PolyATract®, Promega-Serva, D-69042 Heidelberg), isolated. Genomic DNA was isolated using CsCl-Dich Gradient centrifugation followed by proteinase K-Be action and phenolization.

Highly competent bacteria (∼1 × 10⁹ colonies / µg DNA) were found the Inoue et al. (1990) by cultivating the Bacteria obtained at 18 ° C up to an OD of 0.6. The bacts Rien were transferred to a buffer containing MnCl₂ and CaCl₂ and stored with 7% DMSO in liquid nitrogen. The cDNA syn these using the vector primer pXPRS + was carried out with the kit Clonstruct® from USB, Cleveland, Ohio, U.S.A., according to the pre written by the manufacturer. Here the po ly (A) part of the mRNA of WEHI-3 cells to a poly (T) part of the open vector primer, and this was then ßend the first and second strand synthesis performed. Means Terminal transferase became a poly (C) part at the 5 'end of the gene is synthesized and a complementary poly (G) strand by restriction of the 3'-end with BstXI for ligation fen. According to the information from Inoue et al. (1990) transfor with these ligated vectors miert, with a total of about 5 × 10⁵ transformants were. These were amplified once in 500 ml LB medium subsequent isolation of the plasmid DNA using CsCl density gradient centrifugation. An aliquot of the DNA bib thus obtained Liothek was cut uncut on a 1% agarose gel  Sizes separated. With the help of a supercoiled DNA re ferenz (Life Technologies, D-76339 Eggenstein) were plasmids cut out of the gel with insert sizes from 600 to 2,500 bp and cleaned up. The DNA obtained in this way was used again Transfection of highly competent DH5 bacteria as part of the Ex pressure cloning used.

To prepare the phage library from NIH-3T3 mRNA, the Time Saver® cDNA synthesis kit from Pharmacia Biotech, D-79111 Freiburg used according to the manufacturer's instructions. To both Ends of the cDNA were ligated to NotI-EcoRI adapters. The EcoRI Interfaces were made with those of the EcoRI-cut lambda ZapII® phages from Stratagene, La Jolla, CA., U.S.A., left yaws. Then the phages with the packaging kit Gigapack II® from Stratagene packed and according to regulations used by this manufacturer to infect Sure® bacteria. After selection of positive clones with a radioactive probe were inserted together with the plasmid pBluescript® SK by the helper phage R408 out of the lambda phages cut.

The standard gel electrophoresis of DNA was 0.8 up to 1.5% agarose gels. DNA fragments were made in low melting Point Agarose SeaPlaque® GTG® (FMC Bio Products, Rockland, ME., U.S.A.) separated and then cut out. With GELase® (Bio zym Diagnostik GmbH, D-31833 Hess. Oldendorf) was then ß liquefied the agarose according to the manufacturer's instructions. The cleaning was done with the help of Microcon 100® concentrate ren (Amicon GmbH, D-58453 Witten). For the analytical fraction RNA was used in 2.2 M formalin-1.2% agarose gels det. DNA and RNA gels were used for hybridization on Hybond-N® Nylon filter membranes (Amersham-Buchler, D-38110 Braunschweig) using a VacuBlot® apparatus (Pharmacia Biotech, D-79111 Freiburg) blotted. The radioactive detection of certain DNA or  RNA bands on the nylon membranes were made by Markie of the corresponding probes with 32 P-dCTP using the random Primed DNA labeling kits from Boehringer Mannheim. After Wa the hybridized filter membranes according to the stringency according to the regulations of Sambrook et al., In 1989, X-ray films were exposed with these filters. For Fuji Imaging Plates (from Fuji Photo Film Co., Ltd., Japan) with the filters exposed and in a Fuji Phospo imager digitized and evaluated by computer. The DNA sections were sequenced via the chain ab fractionation process according to Sanger et al. (1977) using Di-deoxynucleotides.

The transfection of Cos-1 and Cos-7 cells was carried out with the help the DEAE dextran method (Cullen, 1987). After 30 min incubation the cells with 500 ng DNA / 3.5 cm² culture dish and 5% DEAE-Dex tran, and after 2.5 hours. Incubation with 80 µM chloroquine de instead of the DMSO shock specified in the regulation 3-minute incubation with 15% buffered glycerin leads. As a result, the background of the Differentiation induction by the cos cell supernatants enough. The conditioned Cos supernatants were after 72 hours the harvested and in geometric dilution series with initial 50% supernatant from the murine erythroleukemia cells in 96 micro corrugated sheets added. The induction of differentiation in these Approaches were made by counting the percentage of benzidine-posi tive cells determined after 3 and / or 4 days. In addition, a Stock solution of 10 mg N, N, N ′, N′-tetramethylbenzidine (Sigma Bio chemicals, D-82039 Deisenhofen) in 10 ml of 12% acetic acid sets (NTMB solution). Immediately before staining one was Dilution of 35 µl NTMB solution with 35 µl isopropanol and 5 µl 30% H₂O₂ applied. Depending on the cell density, 5-10 µl of the cell len pipetted into a new microwave and with fresh RPMI 1640 medium filled with 10% FCS to 100 µl. For this purpose 5 µl  the diluted NTMB solution. Between 10 and 30 minutes after that, the benzidine positive cells were percentageized Because of their green color under an inverted microscope in the Mi counted krowells.

For the production of WEHI-3 conditioned media the Cells 3 days beforehand at a density of 2.5 × 10⁴ / ml in RPMI 1640 medium with 10% FCS and the usual other additives sown. After 3 days the cells were at densities between 3.5 × 10⁵ and grown up to 1.2 × 10 her / ml. They were spun down and washed 1 × in RPMI 1640 medium without additives and then in serum-poor medium set to 1 × 10⁶ / ml. After 3 days de the conditioned medium is harvested and after sharp abz centrifugation with an Amicon 10 concentrator concentrated 10 times. These WEHI-3 conditioned media (WCM's) were aliquoted and frozen at -20 ° C. The supernatants were in this form over 3 years with a gradual 2- to 8-fold activity loss durable.

WCM was concentrated 20 to 50 times for fractionation by gel filtration with Sephacryl 300® in a gel bed of 90 × 2.6 cm with PBS as a running buffer and a running speed of 10 ml / min separated into individual fractions and at 280 nm detected. BSA (68 kDa), Chymo served as molecular standards trypsinogen (25 kDa) and cytochrome C (12.5 kDa).

The relative cell count per microwell in 96-well plates was determined determined using the MTT test (Mosmann, 1983). Here will the ability of the cells to measure the yellow tetrazolium salt of 3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-tetrazolium bromide (MTT) into the purple MTT formazan. That ability is bound to the dehydrogenases of active mitochondria, with which ultimately the cell count relative to the number of active mito chondria is determined. The color intensity was with a  Elisa-Reader (SLT, Salzburg) at a test wavelength of 550 nm and a reference wavelength of 690 nm.

The adherence of WEHI-3 cells was checked after incubation with WCM by adhering to the plastic bottom of 96-well plates (Nunc GmbH, 65203 Wiesbaden) tested. This was the supernatant taken from the individual microwells and the bottom of the mic wells rinsed 1 × with fresh medium (RPMI 1640 without additives). After aspirating the medium with the detached cells in the over was fresh RPMI 1640 medium with 10% FCS and 2 mM 1-glow amine added and the MTT test performed. The losge dissolved and previously floating cells in the supernatant pipetted into new microwells and in the same way measured the MTT test.

Results EDA activities in different cell lines

Fig. 1 shows the influence of 4 different WEHI-3 conditioned media on the number of benzidine-positive F4N cells, and thus on the differentiation and hemoglobinization of these cells. The number of benzidine-positive cells when incubated with 1.2% DMSO is shown as a positive control. With DMSO, up to 70% of F4N cells become positive on day 4. With comparable strength of the induction of differentiation with WCM in this experiment, however, the kinetics are different from that with DMSO: Only on day 3 is a differentiation induced by WCM in these cells, which clearly differs from the negative controls. This activity in the WCM, measured on murine Friend Erythroleukemia cells, received the working title "EDA" for Erythroid Differentiation Activity. The strength of the induction of differentiation with the same WCM was subject to stronger fluctuations in the individual experiments, which can be attributed to the willingness to differentiate the erythropoietic cells. Factors of the medium, the FCS and the initial cell density of the erythroleukemia cells were important. A reference WCM was therefore routinely examined as a positive control in such studies. EDA was not only found in supernatants from WEHI-3 cells, but, in lower concentrations, also in those of NIH-3T3 cells, and also in supernatants of the human bone marrow stromal cell line L88 / 5, but only after irradiation and a longer cultivation period ( Fig . 2).

EDA effect on murine and human cells

The factor acting on the mouse cells can therefore be both murine and human. The murine factor in WCM also has a weak effect on human K562 cells, which cannot be induced by DMSO for erythropoietic differentiation ( Fig. 3, especially WCM (C)). The activity labeled EDA is therefore cross-species in both directions.

Biological properties of the activity investigated

The effect of EDA on mouse erythroleukemia cells is not primarily an inhibition of proliferation. Although in many experiments an inhibition of proliferation was of considerable importance at higher concentrations of the factor, experiments such as e.g. B. shown in Fig. 4 with a clear differentiation induction, recognizable by the increase in α-globin mRNA expression, no influence on the proliferation. This differentiation induction was associated with a downmodulation of the c-myb transcript, which was also clearly demonstrated when other cell types such as 32DCl23 were incubated with WCM, and which was also associated with a concentration-dependent extension of the c-myb mRNA half-life. Since a downmodulation of c-myb can be found in many hemopoietic cell systems as part of a differentiation, it is assumed that the erythropoietic induction of EDA differentiation represents only the effect on one of several cell systems. In this sense, the increase in the adherence of WEHI-3 cells that we have proven by their own conditioned medium ( Fig. 5) may be to be seen as an auto-inductive effect in the sense of a differentiation induction. In this context, the increase in the adherence of these myelomonocytic cells means a differentiation step in the direction of macrophages. Based on these findings and assuming that the effects were also caused by EDA, there is a possibility that EDA is a factor which, at least in hemopoietic cells, generally induces differentiation.

Physical and chemical properties of EDA

Some physical and chemical properties are in Ta belle 1 summarized.

Exclusion of individual cytokines as the cause of the EDA effect

In theory, a number of cytokines are responsible for causing the Effects that are considered in the differentiation assay with the mu Pure erythroleukemia cells can be observed. Different Zy tokine could be excluded as the cause. (Tab. 2).

Fractionation of WEHI-3 conditioned media

WEHI-3 conditioned media were fractionated using Sephacryl S300® gel filtration. After the addition of 0.05% BSA as a 20% volume fraction, the individual fractions were incubated with B8 / 3 cells in 5 ml cultures for 4 days. The relative amount of α-globin mRNA formed was then quantified. The main summit in Fig. 6 ran with the small molecular flank of the BSA.

Search for optimal conditions of EDA expression for express ion cloning

WEHI-3 cells were grown to different densities in 250 ml bottles in FCS-containing medium. The larger part was harvested for the preparation of mRNA after reaching the different densities ( FIG. 7), and a smaller part was cultivated under standard conditions for 3 days in order to obtain WCM in serum-poor medium. Fig. 7 shows that the desired densities of about 2.5 × 10⁵ (W3 / 1), 5 × 10⁵ (W3 / 2) and 8 × 10⁵ cells / ml (W3 / 3) were reached after different times. The cells were further incubated at a uniform density of 1 × 10⁶ / ml. The highest EDA activity was found in the W3 / 2 supernatants ( Fig. 7). The RNA of the harvested W3 / 2 cells was therefore further processed for the construction of an expression library. From Fig. 7 it is also clear that at higher concentrations of WCM's there is always an inhibition of differentiation induction. At the same time, an inhibition of proliferation is found for these concentrations. In Fig. 8 for the WCM of W3 / 2 it is shown that the optimal concentration for a differentiation induction in B 8/3 cells by WCM fractions between 66 and 25 kDa is 25%. Although the main peak of EDA was found in this analysis in accordance with the results in Fig. 6 with a molecular size between 60 and 40 kDa, other, similar studies showed clear activities down to molecular sizes of 10 kDa. The existence of different sized molecules with EDA effect, or the aggregation of EDA in different sized aggregates, is one possible explanation.

When testing the optimal culture conditions of WEHI-3 Zel  len for gaining high activity from EDA noticed that the type of primary incubation in 10% FCS is important. When testing 6 different batches of FCS various The manufacturer's EDA activity yield was very low different. With the cheapest FCS, the other Versu che employed. Another important finding was that the Obtaining EDA activity was not possible when in the second Cultivation part (normally 72 h in low serum medium with 1% BSA) use the same medium as in the first part with 10% FCS det (data not shown).

Construction of an expression library with W3 / 2 RNA and the Vector pXPRS +

The unamplified library contained approximately 5 × 10⁵ clones. After amplification and size selection (inserts <600 bp), approximately 500 clones were plated on a round Hybond-N® filter of 14 cm in diameter on LB agar. The clones were removed by filter clap on a second filter to isolate the plasmid DNA. After transfection of this DNA in Cos-1 cells, the Cos-1 supernatant was tested in a batch with B8 / 3 cells. A total of 25,000 clones were screened in this way. In series D there was a positive signal ( Fig. 9). The 520 clones of the D series were divided into groups of approx. 25 each and tested again (D15 series). This was followed by a step with approx. 10 clones (DX series), and finally each clone of the last 10 was tested individually (DY series). The clone DY-8 had an activity 1-2 times lower than the positive control.

Radioactive screening of the pXPRS + library with the clone DY-8

The clone DY-8 showed a size range of approx. 950-750 bp, whereby instead of a sharp band a lubrication of approx. 200 bp was present. Due to the suspicion of an incomplete clone, the gene library was screened radioactively with DY-8 as a probe. A total of 32 positive clones were obtained, the largest being approximately 1,350 bp. This clone was given the designation HA-15/2. It showed a weak differentiation-inducing effect, for which no statistical significance was achieved in 3 experiments ( Fig. 10). Because of its size, it served as a radioactive probe in the further detection work by hybridization (Southern blot, Northern blot, further screening of another gene library), even if it was assumed that it was an incomplete clone.

Creation of a gene library in lambda phages

Assuming that eda's strongest activity is from a 2.2 kbp large mRNA species originate, which are particularly strong in NIH-3T3 cells are expressed (see below), cDNA was extracted from NIH-3T3 rewritten poly (A) + mRNA and on a non-denaturing Gel separated by size. The range from 1,900 to 2,500 bp was cut out of the gel, purified and in lambda Zap II® phage cloned. All eda clones with this ver driving were obtained, but were smaller than 1,500 bp.

Southern blot analyzes

Analyzes of genomic DNA from the spleen of C3H mice showed hybridization with the 1,350 bp probe HA-15/2 (BamHI fragment), in particular after digestion of the DNA with SacI, 4 bands of 6.5, 5.7 , 3.8 and 2.1 kbp. With EcoRI, 2 weak bands at 7.5 and 5.5 kbp were found in the DNA of WEHI-3 cells. It was important to observe that human genomic DNA, obtained from the K562 cell line, also had one band at 7.5 kbp and another at 6.5 kbp after EcoRI digestion ( Fig. 11). Since the washing conditions had been highly stringent (up to 2 × 30 min 0.1 × SSC / 0.1% SDS at 60 ° C.), the murine and the human gene should have some homology. Fig. 11 shows the possibility of extracting the human eda gene from a gene library using a murine radioactive probe.

Investigations of eda gene expression

The investigations were carried out with the approximately 1,350 bp BamHI fragment of HA-15/2 as a probe. A stringent wash (up to 20 min 0.1 × SSC / 0.1% SDS and 60 ° C) shows a number of differently sized and differently hybridizing bands in the Northern blot ( Fig. 12). Individual bands that are larger than 5 kbp differ from cell type to cell type (see below). The strongest band is generally 2,200 bp, other bands are found in different strengths at 1,750 bp, 1,350 bp and 1,200 bp. Fig. 12 shows that all bands hybridize with the entire sample (BamHI fragment of HA-15/2) as well as with the 500 3'-base pairs of this sample, whereas the 200 base pairs at the 5'-end of this sample hybridize only with the large bands over 5,000 bp and with the 2,200 bp band. From this it can be concluded that the large bands contain at least portions of all smaller bands, but that the 2,200 bp band is the only one of the smaller bands that contains the 5'-part of the sample, while the 3'-part of the sample in all bands occurs together. An examination of different mouse strains because of the difference in the bands in the 2 cell types NIH-3T3 and M2-10B4 ( Fig. 12) showed that within a strain in the different tissues all bands are identical, but that the bands of Vary mouse trunk to mouse trunk. NIH-3T3 cells are fibroblasts derived from Swiss mice, while the bone marrow fibroblasts of the M2-10B4 line are derived from B6C3F₁ mice (Lemoine et al., 1988). Similarly, there are numerous restriction differences between the individual mouse strains at the genomic DNA level (data not shown).

Expression patterns of eda in normal mouse tissues

The expression pattern of the various was determined on C3H mice Fabric checked. The strongest expression was found in normal Thymus (adult and fetal (d. 15) about the same) and in the foeta len liver (d. 15). The spleen followed in terms of expression, and the expression in normal bone was significantly weaker mark. A very weak expression, sometimes only on poly (A) + Detectable level, was found in all examined organs and Tissues such as liver, kidney, intestine, brain and placenta. Primary Mast cells, on the other hand, were taken from the bone marrow through 4 weeks Bone marrow cultivation were cultured with IL-3 more positive than normal spleen cells.

Expression patterns in murine cell lines

The expression level in the cell lines was very different Lich, but generally stronger than in the primary tissues. Among the non-malignant cells, the embryonic fibrobla showed line NIH-3T3 a strong expression, while the Fibrobla Line L929 showed almost no expression. A middle class the bone marrow fibroblast line M2-10B4. A very strong expression was found in the T helper cell line TS1-C3, while the EL-4 thymoma cell line showed almost no expression pointed. The myeloid cell lines FDCP-1 and 32DCl23 as well as the Mast cell line 138-8A showed poor expression. The Zelli established from murine malignant hemopoietic tissues In general, expressions were strong to very strong sion. This was the case with the WEHI-3 cells, DA-3, NFS-60 and NFS-61 (all cell lines are derived from murine leukemia)  Lich.

Expression pattern in human cells

With HA-15/2 as a radioactive probe, expression of eda in human cells can be demonstrated, albeit with considerable background. The background relates to a strong co-hybridization of 28S ribosomal RNA, so that 0.2 × SSC / 0.1% SDS must be washed strictly at 20 ° C. for 20 min in order to recognize the decisive bands. We found a transcript at 2.5 kbp in particular in the poly (A) + RNA of Jurkat cells, and such a band is also weakly seen in the total RNA of K562 cells ( Fig. 13). Of the low molecular weight eda mRNA species, a distinct band at 1,100-1,200 bp has only been found in one case of T-CLL using the murine probe (data not shown).

Investigation of eda mRNA expression in murine spleen cells

Spleen cells lose their eda expression within a few hours after in vitro incubation (1 × 10⁶ cells / ml in RPMI 1640 with 10% FCS and the usual additives). When anti-T cell receptor antibody or concanavalin A ( Fig. 14) or TPA is added, the 2.2 kbp band and the larger bands are slightly increased within 1 hour after 4 hours however, these transcripts have already been reduced and there is an accumulation of eda degradation products (<400 bp) without stabilization of the normal transcripts. These effects largely disappear within 24-28 hours.

In contrast, eda mRNA in spleen cells was stabilized in vitro when a 3-day mixed spleen cell reaction with 1 × 10⁶ unexposed and untreated CBA and CBL cells / ml was carried out ( Fig. 15). In contrast, if the CBA spleen cells had previously been irradiated with 15 Gy, there was again no stabilization of the mRNA, just as little as if CBL spleen cells were cultivated in vitro on their own.

The involvement of eda in the allogeneic reaction, interestingly only when stimulated by unirradiated cells ( Fig. 15), was confirmed in an in vivo model of acute graft versus host (GvH) disease. If (CBA × CBL) F₁ hybrids after 9 Gy whole body irradiation 5 × 10⁷ CBL spleen cells are injected, severe GvH disease develops. As part of this disease, we found an approximately 7-fold increase in eda expression in the spleen of the recipient animals on the 6th day after the transplant in comparison to the controls in which the eda expression fell ( Fig. 16). This response was limited to the spleen, probably because in the other organs examined the percentage of inflammatory cells involved in the GvH disease, based on all cells, was too low to increase eda expression in the Northern blot close.

Sequence analysis by eda

The DNA sequence of eda, or of the different cDNA species, proved to be difficult to analyze, especially because of the numerous repeat structures and AT-rich sections. A consensus sequence was derived from various clones of the pXPRS + ® and Lambda Zap II® libraries ( Fig. 17), which is most likely to be part of the 2.2 kbp cDNA. Fig. 18 shows the assumed structure of the 2.2 kbp sequence, which could not yet be fully analyzed. The sections marked with thick lines are secured in terms of their relative assignment. However, it is not certain whether the approx. 500 bp that are still missing are in the beginning of the sequence, as indicated in Fig. 18. As a result, an open reading frame has not yet been found. Figure 19 shows the sequence of the DY-8 clone. This sequence contains an open reading frame, but the activity of this clone when transfected in Cos cells is not strong enough to assume that the real 5 'end has already been detected.

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Table 1

Percentage decrease in EDA activity

Table 2

Cytokines with no differentiation-inducing effect on mouse erythroleukemia cells

Table 3

Repeat structures in the presumptive consensus partial sequence of the 2,200 bp eda cDNA

SEQUENCE LOG

Claims (32)

1. Protein with differentiation-inducing activity with the following properties:

• a) isolatable from murine myelomonocytic leukemic cell lines;
• b) isolatable from irradiated human bone marrow current cells;
• c) induces differentiation in friend erythroleukemia cell lines with hemoglobin formation;
• d) with a molecular weight in the range of about 10-60 kDa;
• e) inducible by a serum factor occurring in fetal calf serum;
• f) with an expression of the associated mRNA in primary cells from thymus, fetal liver, adult spleen or bone marrow;
• g) with a stable expression of the associated mRNA in vitro if an allogeneic spleen cell reaction with unirradiated, not pretreated spleen cells of the mouse strains CBA and C57Bl6 is carried out;
• h) with characteristic repeat structures in the cDNA coding for the protein;
• i) with AT-rich sections in the cDNA coding for the protein;
• k) with associated mRNA species of different sizes, which consist of the same 3'-regions but different 5'-regions.

2. Protein according to claim 1, characterized, that the murine myelomonocytic leukemic cell line WEHI-3, ATCC is TIB68 and the Friend Erythroleukemia cell line F4N or B8 / 3 and the irradiated human bone marrow current cell line L 88/5 (DSM ACC 2056). 3. Protein according to claim 1, characterized, that one or more of the repeat shown in Table 3 sequences or with these repeat sequences conditions hybridizing repeat sequences in the for DNA encoding the protein of claim 1 or 2 gene. 4. Protein according to one or more of the preceding An claims, characterized, that it is from human cells, mouse cells or the culture is isolated from human or mouse cell lines. 5. Protein according to one or more of the preceding An claims,  characterized, that it has a partial amino acid sequence that is from a hybridizing with the cDNA of SEQ ID NO: 1 or NO: 2 DNA is encoded. 6. Protein according to one or more of the preceding An claims, characterized, that it has a partial amino acid sequence that is from a with the cDNA according to SEQ ID NO: 1 or NO: 2 under stringent Conditions hybridizing DNA is encoded. 7. Protein according to one or more of the preceding An claims, characterized, that parts, analogs and derivatives of the protein as well as Fu sionsproteins are included. 8. Protein according to one or more of the preceding Claims in an essentially purified, native form. 9. Protein according to one or more of the preceding Claims in essentially recombinant form. 10. Protein according to one or more of the preceding Claims with differentiation-inducing activity for erythropoietic cells. 11. Protein according to claim 1, characterized, that there is an erythropoiesis-inducing effect on the human leukemia cell line K 562 (ATCC No. CRL243) having. 12. DNA fragment according to SEQ ID NO: 1 or NO: 2, parts, derivatives and analogues thereof.   13. DNA fragments, parts, analogs and derivatives thereof, the hybridize with the cDNA according to SEQ ID NO: 1 or NO: 2. 14. DNA fragments, parts, analogs and derivatives thereof, the with the cDNA according to SEQ ID NO: 1 or NO: 2 under stringent Hybridize conditions. 15. DNA fragment according to one or more of the preceding Expectations, characterized, that it encodes at least part of a polypeptide, which is the activity of human or murine Protein with differentiation inducing activity for erythropoietic cells according to one or more of the previous claims. 16. recombinant vector, characterized, that it contains a DNA sequence that a gene or a DNA fragment corresponds to that for a protein with differen ornament inducing activity for erythropoietic Coded cells according to one of the preceding claims. 17. Recombinant vector according to one or more of the previous claims, characterized, that it is from a bacterial plasmid, a bacteriopha gene or derived from a viral vector. 18. Host cell transformed from a vector to or several of the preceding claims. 19. host cell according to claim 18, characterized, that they are a prokaryotic cell or a eukaryotic cell is.   20. Method for producing a DNA fragment according to a or more of the preceding claims, characterized, that it is the screening of a human or murine cDNA clone bank using a DNA fragment of a DNA, for a murine or human protein with diffe coding inducing activity, as a probe includes. 21. A monoclonal or polyclonal antibody against a protein with differentiation-inducing activity for erythropoietic cells, a part, a derivative or an analogue thereof according to one or more of the previous claims is directed. 22. Therapeutic, diagnostic or experimental use cash, characterized, that there is at least one nucleic acid in contains an effective amount associated with a gene or hybridized a part of it that for a protein with Differentiation-inducing activity for erythropoeti cells according to one or more of the preceding Claims encoded. 23. Means according to one or more of the preceding claims che, characterized in that it is at least as an active substance contains a nucleic acid which (a) has the for a protein Differentiation-inducing activity for erythropoeti nucleotide sequence coding for cells, (b) a part of these, (c) one with a nucleic acid from (a) and / or (b) hybridizing under stringent conditions Nucleotide sequence or (d) one to a nucleotide sequence from (a), (b) and / or (c) complementary nucleotide sequence includes.   24. Means according to one or more of the preceding an claims, characterized, that the nucleic acid is an optionally modified DNA is. 25. Means according to one or more of the preceding an claims, characterized, that the nucleic acid is an optionally modified RNA is. 26. therapeutic agent, characterized, that it's a protein, an analog, derivative, or parts of which according to one or more of the preceding an say together with usual carrier and / or auxiliary fen contains in an effective amount. 27. Use of an agent according to one or more of the previous claims as a molecular probe in the Diagnosis. 28. Use of an agent according to one or more of the previous claims as an antisense nucleic acid Inhibition of gene expression. 29. Process for transforming a prokaryotic or Eukaryotic cell using one for a protein DNA encoding erythropoiesis inducing activity, a part, a derivative or an analogue thereof. 30. Fusion protein with an amino acid sequence that is in its All or part of the amino acid sequence of human or murine protein with differentiation inducing activity for erythropoietic cells  one or more of the preceding claims and one Part of a prokaryotic or eukaryotic protein consists. 31. Synthetic protein with differentiation-inducing Activity for erythropoietic cells after one or several of the preceding claims with an amino acid sequence encoded by a DNA sequence that with the DNA sequence according to SEQ ID NO: 1 or NO: 2 at least hybridized under stringent conditions. 32. Use of a protein according to one or more of the preceding claims or the inhibitors of this Protein for the treatment of diseases in which a local or systemic over or under production of this Protein on disease development or its course has an influence.