K562 cells are human erythroid cells that synthesize embryonic and fetal globins but not adult ,-globin. A cloned 0-globin gene was isolated from K562 cells and transfected into HeLa cells. The RNA transcripts produced were comparable in both amount and size to those obtained with a normal ,-globin gene.
Specific globin mRNA accumulation was quantitated in several lines of K562 cells in the absence and the presence of hemin. Using specific cloned DNA probes, the amounts of zeta, alpha, epsilon and gamma mRNAs were shown to be increased 2–3-fold in the presence of 20 microM hemin. No delta- or beta-globin mRNAs were detectable in any of the lines. In one line, Bos, there was a marked decrease in epsilon-globin mRNA, which increased with hemin, although still to much lower levels than in the other lines. The decreased epsilon-globin mRNA accumulation in Bos is shown to be due to decreased epsilon-globin gene transcription.
K562 cells are induced by hemin to produce gamma and epsilon globin but not beta globin, although the beta globin gene is intact, and when isolated is expressed in a transient expression assay (1, 2). We have previously shown that an epsilon globin gene transferred into K562 cells is expressed and inducible (3). In this paper, we report the stable transfer of a sickle or betaS globin gene into K562 cells. Thirty-six different transformed lines were tested; 24 of 36 lines contained an intact betaS globin gene. However, using S1 nuclease, Dot blot, and Northern blotting analyses, none of these lines showed beta globin mRNA expression. These results indicate that trans acting factors are responsible for the lack of expression of the beta globin gene in K562 cells.
To study the expression of globin genes in human cells, human e-globin genes were transferred into a K562 cell line, Bos, which synthesizes very low amounts of E-globin mRNA. A plasmid (pSV2neo-e) containing a complete E-globin gene and 2 kilobases (kb) of 5' flanking DNA as well as a neomycin-resistance gene and a simian virus 40 origin of replication was transfected into Bos cells; the compound G418, a neomycin analogue, was used to select transformed cells. The presence of unique bands by DNA restriction analysis shows that 11 of 14 of the G418-resistant clones have at least one copy of an integrated E-globin gene. RNA expression measured by RNA blotting shows significantly more E-globin mRNA sequences than in untransfected Bos cells in 10 of 11 lines; in most lines, E-globin mRNA was additionally increased in the presence of hemin. In two lines, E-globin mRNA expression with hemin was comparable to that of a high E-globin producing cell line, K562 clone 2. The one G418-resistant line without E-globin genes had no E-mRNA expression. The high E-mRNA expression in several of the lines suggests that exogenous E-globin genes with only 2-kb 5' flanking DNA may be sufficient to be appropriately expressed in these homologous erythroid cells. These results have implications for the potential success of transfer of normal human genes to human bone marrow cells as an approach to the treatment of inherited anemias.
Over the past five years, several new defects in the beta-thalassemias have been described from this laboratory using both restriction enzyme and sequencing analyses of cloned beta-thalassemia genes. The enzyme HphI has been shown to recognize a single nucleotide change at the 5' end of beta-IVS 2, and, using restriction enzyme analysis, demonstrated for the first time a specific defect associated with beta(0)-thalassemia. Cloning and sequencing of a beta-thalassemia gene have identified a single base change within IVS 2 at a position 705 nucleotides from the 5' end of IVS 2 that results in a beta(0)-thalassemia phenotype; no normal splicing occurs in this gene despite the fact that both the 5' and 3' ends of IVS 2 are unchanged. A unique and strong cryptic 3' acceptor splice site present in the normal gene at a position 580 nucleotides from the 5' end is used extensively in the mutant gene. Studies of this gene have indicated that there are sequences within IVS that are responsible for optimal expression of this gene; changes in these sequences can lead to markedly abnormal patterns of splicing. In addition, beta-globin gene expression has been evaluated in human erythroleukemia cells, K562 cells, and, although stable transformants with integrated beta-globin genes have been obtained, none of these transformants expressed the added beta-globin genes. This is presumably due to trans-acting factors or distal cis-acting effects that suppress the expression of these added beta-globin genes. In addition, a low epsilon-producing cell line, Bos cells, was used as a recipient for an exogenous epsilon-globin gene. A neomycin resistance gene was cotransfected into these cells, and a neomycin analogue (G418) was used to select cells containing both the neomycin resistance and epsilon-globin genes. Using Southern blotting, 10 of 11 stably transformed G418-resistant lines, which contain intact epsilon-globin genes, express epsilon-globin mRNA at much higher levels than the Bos cells into which they were transfected. Two of these lines express the epsilon-globin genes at a level comparable to that of wild-type K562 cells. These results indicate that the transfer and expression of human globin genes in human erythroid cells is feasible, and can occur at a high level.(ABSTRACT TRUNCATED AT 400 WORDS)
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