High level accumulation of an aberrantly spliced human DHFR RNA species.

Cells transduced with either of two human DHFR minigenes express an RNA product which is considerably shorter than what would be predicted from the size of an unspliced transcript expressed from its DNA template. RNA blotting analysis has shown that this short transcript accumulated to exceedingly high levels which were comparable to the levels reached by the highly abundant endogenous actin mRNA, or MoMLV RNA expressed in chronically infected cells. RNA blotting, RNase mapping, primer extension, RT-PCR, and sequencing have shown that this highly expressed transcript, termed TBN, is a spliced RNA product which utilizes cryptic splice signals present in the normally spliced DHFR mRNA. Subcloning experiments have demonstrated that all the information required for the generation and high level accumulation of the TBN transcripts is encoded in the 1.6 kb DHFR DNA minigene. TBN transcripts were generated with comparable efficiency from DNA templates containing either the human ADA or the early SV40 promoters. Since neither the ADA nor the SV40 promoter are considered to be particularly "strong" promoters, this observation argues that initiation of transcription is not the rate limiting step in determining the amount of the TBN transcripts which accumulate in the cell. Insertion of a foreign sequence into the DHFR DNA minigene led to the expression and high level accumulation of a chimeric transcript, suggesting that the unusual properties of this expression system may be used for high level expression of foreign sequences. These observations offer new insights into the mechanisms which control the accumulation of translatable mRNA in the cell, and have potentially important implications for experiments involving optimization of gene expression for gene therapy applications.

Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction.

A double-copy Moloney leukemia virus-based retroviral construct containing both the NeoR gene and a mutant human dihydrofolate reductase (DHFR) cDNA (Ser31 mutant) was used to transduce NIH 3T3 and mouse bone marrow (BM) progenitor cells. This resulted in increased resistance of these cells to methotrexate (MTX). The transduced BM progenitor cells were returned to lethally irradiated mice. The recipients transplanted with marrow cells infected with the recombinant virus showed protection from lethal MTX toxicity as compared with mock-infected animals. Evidence for integration of the proviral DNA was obtained by amplification of proviral DNA by polymerase chain reaction (PCR) and Southern analysis. Sequencing a portion of the PCR-amplified human DHFR cDNA showed the presence of the mutation. These studies with the human Ser31 mutant DHFR cDNA gave results comparable with those obtained with the mutant murine DHFR cDNA (Leu to Arg22) in developing MTX-resistant BM. The Ser31 mutant human DHFR cDNA is currently being tested for infection of human CD34+ human BM and peripheral blood stem cells in vitro.

Long-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a mutant dihydrofolate reductase.

A double-copy Moloney murine leukemia virus-based retroviral construct containing both the NEOr gene and a mutated dihydrofolate reductase cDNA (Leu 22-->Arg) was used to infect mouse bone marrow cells. The infected mouse marrow was returned to lethally irradiated mice. Primary, secondary, and even tertiary recipients transplanted with bone marrow cells infected with the recombinant virus showed protection from lethal methotrexate toxicity. The viral construct containing a SV-40 promoter in the U3 region of the 3' long terminal repeat appeared to be more effective than a similar construct containing the adenosine deaminase promoter, although both afforded protection. Evidence for integration into blood cells of both the NEOr gene and the mutated dihydrofolate reductase gene was obtained by polymerase chain reaction; sequencing of the amplified dihydrofolate reductase cDNA showed the presence of the point mutation. These results indicate that early hematopoietic progenitor cells in the mouse can be successfully transduced with a drug resistance gene.

Retroviral gene transfer of epidermal growth factor receptor into HL60 cells results in a partial block of retinoic acid-induced granulocytic differentiation.

HL60 cells are devoid of endogenous epidermal growth factor receptor (EGFR). They respond to retinoic acid and undergo terminal granulocytic differentiation. EGFR complementary DNA was introduced into HL60 cells by retroviral gene transfer. Scatchard plot showed that the binding characteristics are identical to those of A431 cells. HL60-EGFR cells were estimated to express 34,000 EGFR/cell (Kd = 5 nM). The tyrosine phosphorylation upon ligand binding is the first step of signal transduction. The dominant phosphotyrosyl proteins in epidermal growth factor-stimulated HL60-EGFR cells include a 170 kDa protein (EGFR itself), and 125 and 53 kDa proteins. The EGFR signal results in the induction of 92 kDa gelatinase/matrix metalloproteinase in HL60-EGFR cells, thereby providing evidence of the function of the exogenous EGFR and a semiquantitative measure of the EGFR signal. These HL60-EGFR cells offer a unique opportunity to examine the potentially important role of EGFR (c-erbB) in maintaining homeostasis between self-renewal and differentiation. c-erbB has been shown to play a physiological role in the self-renewal of the very early avian stem cells which do express EGFR. The v-erbB (double truncated EGFR) has been shown to cause avian erythroblastosis. We found that these HL60-EGFR cells responded to retinoic acid differently from the HL60-control cells. A partial block of only 45% granulocytic differentiation and concomitant proliferation was noted, consistent with a shift of balance between self-renewal and differentiation toward the former.

Expression of functional epidermal growth factor receptors in a human hematopoietic cell line.

To test the feasibility of using the human epidermal growth factor receptor (EGFR) as a model for growth factor receptor action in human hematopoietic cells, we infected Burkitt lymphoma cells (Namalwa) with a recombinant amphotrophic retrovirus containing a thymidine kinase promoter-driven human EGFR complementary DNA and the neomycin resistance gene. Neomycin-resistant cells expressing surface EGFR were selected by cell sorting using anti-EGFR monoclonal antibody 225. The selected cells expressed a Mr 170,000 protein immunoprecipitated by monoclonal antibody 225 and apparently identical to EGFR from A431 carcinoma cells. Infected Namalwa cells expressed 42,000 epidermal growth factor (EGF) binding sites/cell, and Scatchard analysis showed two affinities (Kd approximately 5 nM and approximately 0.5 nM). EGFR autophosphorylation was detected using antiphosphotyrosine antibodies after 5 min exposure to EGF. EGF binding induced rapid EGFR internalization (t1/2 = 9 min) and mobilization of transferrin receptors to the cell surface within 1 min. In fetal bovine serum-containing and serum-free cultures, EGF did not stimulate Namalwa cell proliferation. However, in the presence of 1.25% dimethyl sulfoxide (DMSO), EGF caused a dose-dependent increase in DNA synthesis. DMSO induced a cell cycle block in G1, which was partially reversed by EGF. DMSO induced changes in some B-cell markers suggesting cellular differentiation and increased surface EGF receptor number. Cells grown in DMSO and EGF were established as an EGF-dependent cell line for greater than 12 weeks, whereas cells grown in DMSO without EGF died within 1-2 weeks. Namalwa cells expressing EGFR demonstrated more rapid tumor growth in athymic mice. These studies demonstrate expression of functional EGFR mediating early biochemical and growth responses in a human hematopoietic cell, and indicate that EGFR can be used as an effective model in human hematopoietic cells. Results using DMSO are consistent with studies in other human leukemia cells indicating that agents inducing differentiation can restore growth factor dependence in previously factor-independent leukemia cells.