Hormone associated therapy of leukemia: reflections.
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Animals↗
Biomedical subjects
W D Hankins
Publications and source records attributed to W D Hankins.
In vitro retroviral transfer of ras genes to single hemopoietic progenitors.
Recent studies have shown that retroviruses can serve as efficient vectors of exogenous genes that can be inserted and expressed in a variety of mammalian cell types. Several investigators have exposed total bone marrow populations to retroviruses in vitro and have demonstrated the presence of exogenous genes after inoculation into irradiated mice. Our approach was to identify individual pluripotent hemopoietic progenitors in vitro and to use these single cells as targets for retroviral gene transfer. This approach was made possible by our previous identification of in vitro colonies containing pluripotent, undifferentiated blast cells with very high secondary replating efficiencies. By using a monoclonal antibody to detect the product of the transferred gene, we were able to document infection of single multipotent cells and to quantitate the percentage of the progeny cells that expressed the transferred gene. Specifically, individual blast cells were obtained by micromanipulation, exposed to Harvey sarcoma virus, and ras gene expression was detected by immunofluorescence in individual colonies. A variety of types of p21-positive colonies were seen, including a macrophage (m)-neutrophil (n)-erythroid (E)-mast cell (mast)-megakaryocyte (M) colony, an mEmastM colony, an nmmast colony, mnE colonies, mn colonies, and m colonies. These results demonstrated that multipotent progenitors were recipients of exogenous genes and that these genes were expressed in the differentiated progeny. Initial experiments failed to demonstrate that the cells in the infected colonies were transformed. Retroviral infection of isolated blast cells may provide a unique method for studies of the effects of a variety of genes, including oncogenes, in hemopoietic cells.
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Treatment of a fatal transplantable erythroleukemia by procedures that lower endogenous erythropoietin.
The in vitro growth of primary erythroleukemia cells has been examined in the presence and absence of the hormone erythropoietin (EPO). Although these leukemic cells had previously been considered to be hormone-independent, addition of EPO was found to be essential for maximum growth in culture. Erythroid colonies that grew in the presence of EPO were leukemogenic when returned to mice. Influence of EPO on the in vivo growth of leukemic cells was indicated by our findings that administration of the hormone caused a more severe leukemia and rapid death, and transfusion of red blood cells, which lowers endogenous EPO, led to decreased spleen size and increased survival of leukemic mice. We suggest from our results that hormone-associated therapy might be efficacious in the treatment of this and, perhaps, other leukemias.
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Purification of murine erythropoietin produced in serum-free cultures of erythroleukemia cells.
We previously documented that several erythroleukemia cell lines released factors that stimulated erythropoiesis in vivo and in vitro. A simple five-step scheme has been devised that allows purification of this erythropoietic activity to apparent homogeneity. The methods employed included lectin affinity chromatography (wheat germ agglutinin), gel filtration (ultro gel ACA44), ion exchange, hydroxylapatite, and high performance liquid chromatography. Following polyacrylamide gel electrophoresis, biologic activity was recovered in an area corresponding to a molecular weight of 35,000 daltons. Silver staining of a polyacrylamide gel after electrophoresis of our most purified preparation revealed a single band at 35,000 daltons.
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Erythropoietin, an autocrine regulator? Serum-free production of erythropoietin by cloned erythroid cell lines.
Production of lymphoid and myeloid growth regulatory factors by hematopoietic cells is well documented. On the other hand, the major site of production of erythropoietin (Epo), which regulates physiologic red blood cell development, is thought to be the kidney. Here we report the isolation of multiple erythroleukemia cell lines that produce erythropoietic factors and present extensive biological, immunologic, and biochemical evidence to document that the active agent is Epo. The erythropoietic activity was neutralized by Epo antiserum and exhibited physical properties indistinguishable from those of human and sheep Epo. Positive lines produced between 0.1 and 1.5 U/mL of Epo, which stimulated erythropoiesis in vivo and in vitro in nine biological assays. Twenty sublines derived from single cells were inducible for hemoglobin and spectrin synthesis. All the sublines produced Epo. Production of the hormone continued when the cells were seeded in the absence of serum. Our finding that multiple independent isolates produce Epo raises the possibility that Epo production by erythroid precursors may play a role in normal erythropoiesis or, alternatively, that Epo gene activation may be a relatively common occurrence that contributes to, or is associated with, certain forms of virus-induced leukemias.
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Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo.
Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.
2-Acetylaminofluorene↗
Rapid determination of DNA synthesis in adherent cells grown in microtiter plates.
A specific, rapid, and economical method for measuring the extent of DNA synthesis in adherent rat hepatoma H4-II-E cells grown in 96-well microtiter plates is described. The adherent cells were pulsed for 1 h with [methyl-3H]thymidine, released from the substratum by trypsinization, and collected on fiberglass filters with a MASH II cell harvester. The amount of radioactivity incorporated was directly proportional to the number of cells per well. Growth curves generated by measuring [methyl-3H]thymidine incorporation and counting the number of cells per well were identical. Experiments with inhibitors of DNA, protein, and RNA synthesis demonstrated that this method selectively measured DNA synthesis. In addition, [3H]thymidine uptake showed excellent correlation with autoradiographic assessment of DNA synthesis. This specific and sensitive method for determining DNA synthesis in microtiter cultures should facilitate studies of effects of various growth-controlling agents on epithelial, fibroblastic, and other cells which grow as adherent cells in culture.
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Induction of the early stages of Friend erythroleukemia with helper-free Friend spleen focus-forming virus.
The polycythemia-inducing strain of Friend virus (FV-P) causes a multistage erythroleukemia in susceptible mice. FV-P is a complex of two viruses, a replication-competent virus [Friend murine leukemia virus (F-MuLV)] and a replication-defective spleen focus-forming virus (SFFVp). We have addressed directly the role of SFFVp in the induction of the early stages of Friend disease by constructing stocks of SFFVp free of detectable F-MuLV, using a recently described retroviral helper-cell line. These preparations are capable of inducing erythroid bursts (vBFU-E) whose inducibility, kinetics, and responsiveness to erythropoietin suggest that they are very similar, if not identical, to the vBFU-E induced by FV-P. Single injections of helper-free SFFVp had no apparent effects in vivo, although the addition of exogenous helper virus to the inoculum resulted in the induction of classic Friend disease. Increasing the effective titer by giving mice five daily virus injections resulted in the induction of splenomegaly and a large increase in the number of erythroid colony-forming units that were independent of erythropoietin. When the injections were discontinued, the spleens regressed and all the mice survived. When the injections were continued, all the mice died within 25 days of the first injection. These results demonstrate that SFFVp alone can alter the growth characteristics of erythroid progenitors and is directly responsible for the induction of vBFU-E in vitro and the erythroid hyperplasia in vivo. They also demonstrate that the initial polyclonal stage of Friend disease is reversible and can be reproduced by using preparations of SFFVp free of detectable F-MuLV.
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Sequence comparisons of the anemia- and polycythemia-inducing strains of Friend spleen focus-forming virus.
A nucleotide sequence analysis carried out on the envelope gene of the anemia-inducing strain of the Friend spleen focus-forming virus (F-SFFVA) reveals that its product has some unique features in common with previously described polycythemia-inducing strains of F-SFFV (F-SFFVP). (i) It contains an amino terminus that is highly related to the gp70 of mink cell focus-inducing viruses, (ii) it is a fusion protein containing the amino terminus of gp70 and the carboxy terminus of p15E, and (iii) it lacks the R-peptide normally found at the carboxy end of the p15E region. Although the envelope genes of F-SFFVA and F-SFFVP are quite similar overall, they do show sequence variation, particularly at the 3' end in the p15E-related region. These variations may contribute to previously observed differences in the response of F-SFFVP- and F-SFFVA-infected erythroid cells to regulatory hormone or to differences in the way the envelope glycoproteins are processed. The long terminal repeat regions of F-SFFVA and the Lilly-Steeves strain of F-SFFVP were also sequenced and compared with each other and with a previously published sequence of another F-SFFVP long terminal repeat. The sequences were found to be reasonably similar to each other but different from their ecotropic parent, Friend murine leukemia virus, as a result of a deletion of one copy of the direct tandem repeat in the enhancer regions. The observation that all SFFVS have this common change in the long terminal repeat enhancer region raises the possibility that it is required for pathogenicity.
Amino Acid Sequence↗
Roles of helper and defective retroviral genomes in murine erythroleukemia: studies of spleen focus-forming virus in the absence of helper.
Retroviruses that cause acute oncogenesis are generally complexes of a replication-competent helper virus and a replication-defective component. However, the pure defective components have not been previously available. We prepared the defective spleen focus-forming virus component of Rauscher erythroleukemia virus (R-SFFV) by transfecting a colinear R-SFFV DNA clone into a retroviral packaging cell line (psi 2 cells). The transfected cells released virus (psi 2/SFFV) that was free of helper virus and that induced erythropoietin-dependent erythroid burst formation in bone marrow cultures. When injected into normal adult NIH/Swiss mice in moderate doses, psi 2/SFFV caused a rapid splenic erythroblastosis that regressed. Extensive erythroblastosis could be maintained by repeated injections of psi 2/SFFV into anemic mice or by the addition of a helper virus. We conclude that R-SFFV alone causes proliferation but not immortalization of a population of erythroblasts that is normally replenished from a precursor stem cell pool. Because these precursor cells are inefficiently infected, a single moderate inoculum of psi 2/SFFV causes a wave of erythroblastosis. The properties of the proliferating erythroblasts are substantially determined by the R-SFFV viral component.
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A murine recombinant retrovirus containing the src oncogene transforms erythroid precursor cells in vitro.
A murine retrovirus (MRSV) containing the src gene of Rous sarcoma virus has been shown to cause an erythroproliferative disease in mice (S. M. Anderson and E. M. Scolnick, J. Virol. 46:594-605, 1983). We now demonstrate that this same virus can transform erythroid progenitor cells in vitro. Infection of fetal liver cells or spleen and bone marrow cells from phenylhydrazine-treated adult mice gave rise to colonies of erythroid cells which grew in methylcellulose under conditions not favorable for the growth of normal erythroid cells. The presence of pp60src in the transformed erythroid cells was demonstrated by an immune complex protein kinase assay. The time course of appearance and subsequent differentiation of erythroid colonies indicated that the target cell for MRSV was a 6- to 8-day burst-forming unit. Differentiation of the erythroid progenitors was not blocked by the presence of pp60src, and the cells retained sensitivity to the hormone erythropoietin. In fact, the transformed cells exhibited increased hormone sensitivity since the number, the size, and the extent of hemoglobinization of the colonies were all increased by the addition of small amounts of erythropoietin. MRSV was not susceptible to restriction by the Fv-2 locus, as MRSV could transform hematopoietic cells from C57BL/6 mice. These results indicate that (i) the erythroid proliferation observed in vivo is caused by a direct effect of MRSV on erythroid progenitors and (ii) the transformed erythroid precursors acquire a growth advantage over uninfected cells without losing the ability to differentiate and respond to physiologic regulators.
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Expression of xenotropic-like env RNA sequences in normal DBA/2 and NZB mouse tissues.
Using a DNA probe prepared from cloned env gene sequences of Friend spleen focus-forming viruses, we detected the differential expression of multiple RNA species in uninfected DBA/2 fibroblasts and in various tissues from adult DBA/2 and NZB mice. The size of the major RNA species detected was estimated to be 24S . The 24S RNA species was enriched in polyadenylate-selected preparations and thus may represent a message for endogenous viral envelope glycoproteins. The viral origin of the 24S RNA was further characterized by its hybridization to DNA probes containing the long terminal repeats of Harvey murine sarcoma virus, mouse mammary tumor virus, or the U3 region of an endogenous xenotropic virus. Although the env-related 24S RNA failed to react with either Harvey murine sarcoma virus or mouse mammary tumor virus long terminal repeat probes, it hybridized well with the xenotropic virus long terminal repeat probe. Therefore, it is likely that the RNA detected with the Friend spleen focus-forming virus env probe reflects transcription of xenotropic envelope sequences in uninfected tissues. Our finding that the level of 24S RNA varied in different organs indicated some tissue specificity in the expression of these xenotropic-like env proteins.
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Modification of erythropoiesis and hormone sensitivity by RNA tumor viruses.
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Animals↗
Cas spleen focus-forming virus. II. Further biological and biochemical characterization.
A new isolate of a murine erythroblastosis-inducing spleen focus-forming virus (Cas SFFV), derived from the wild mouse ecotropic murine leukemia virus Cas-Br-M, was further characterized after the production of a nonproducer cell line. When rescued from the nonproducer cells with a helper murine leukemia virus, the Cas SFFV induced rapid splenic enlargement and focus formation when inoculated into adult NFS/N mice. The Cas SFFV nonproducer cell line was also utilized to compare the envelope-related glycoprotein of Cas SFFV with gp52s from three strains of Friend SFFV. Cas SFFV was found to encode a 50,500-dalton glycoprotein (gp50) distinct in size to the envelope-related glycoproteins of the Friend SFFVs. The Cas SFFV was also compared in RNA blot hybridization studies. The genomic viral RNA of Cas SFFV was found to be slightly larger than two polycythemia-inducing strains of Friend SFFV and markedly larger than the anemia-inducing strain. Further comparisons between the SFFVs were made by examining their transforming capabilities in an in vitro erythroid burst assay. The erythroid bursts induced by Cas SFFV and the anemia-inducing strain of Friend SFFV showed similarities in their erythropoietin requirements. This study supports our recent observations that Cas SFFV is biologically similar to the anemia-inducing strain of Friend SFFV yet biochemically distinct from all Friend SFFVs.
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Increased erythropoietin sensitivity after in vitro transformation of hematopoietic precursors by RNA tumor viruses.
Two strains of Friend virus differ in their in vivo actions in that one strain induces anemia (FVA), while the other induces polycythemia (FVP). This study characterizes differences in the in vitro effects of these viruses on hematopoietic cells of (BALB/c x DBA/2)F1 mice. Both variants induced erythroid bursts that proliferated and differentiated without added erythropoietin (EPO). However, while the bursts induced by FVP were well "hemoglobinized" (i.e., most cells contained hemoglobin), the cells of FVA-induced bursts contained little or no hemoglobin. The nonhemoglobinized bursts, induced by FVA, were established to be erythroid by cytochemistry, electron microscopy, and hormone sensitivity. FVA-induced cells appeared to be hypersensitive to EPO, since small concentrations of the hormone produced marked increases in hemoglobin production--even when the hormone was added to the culture 3 days post infection. Time-lapse photography documented that EPO stimulated hemoglobin synthesis in virally transformed cells rather than uninfected erythroid precursors. This observation of FVA-induced hypersensitivity prompted the reexamination of the hormone requirements of FVP-induced bursts--previously considered to be EPO-independent. Reduction of the serum in the cultures allowed the demonstration that FVP-induced erythroid cells also were hypersensitive to EPO. Thus FVA and FVP can be readily distinguished in vitro by the relative EPO sensitivity of virus-induced bursts. From these findings, a hypothesis is drawn: i.e., oncogenic transformation may result from increased sensitivity of progenitor cells for natural, physiologic regulators, and transformation is not necessarily accompanied by a block in differentiation. In addition, since hypersensitive virus-induced bursts could be recognized and picked from the cultures, these studies provide a method for obtaining highly purified erythroid precursors for the study of the regulation of terminal differentiation by EPO and other regulatory factors.
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Transformation of adult and fetal hemopoietic tissues with RNA tumor viruses.
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Animals↗
Specific differentiation events induced by erythropoietin in cells infected in vitro with the anemia strain of Friend virus.
Mouse bone marrow cells infected in vitro with the anemia strain of Friend leukemia virus from large clusters (bursts) of erythroblasts after 5 days in culture in methylcellulose medium. Two types of erythroblast populations can be isolated from bursts of infected cells by manipulation of the culture conditions. One type of erythroblast, which is obtained when erythropoietin (EP) is added to the culture, has proliferated and undergoes differentiation to become an erythrocyte. The second type of erythroblast, which is obtained when no EP is added to the culture, is the product of extensive proliferation, but it fails to undergo the terminal stages of erythroblast differentiation. Comparisons of these two types of erythroblasts demonstrate that specific EP effects include changes in the nucleus, cytoplasm, and membrane of the treated cells. Those events of erythroid differentiation shown to be directed by EP were extrusion of the nucleus from the erythroblast, induction of uroporphyrinogen I synthetase activity, increased iron incorporation into protoporphyrin, synthesis of alpha- and beta-globin polypeptides due largely to increased mRNA production, and synthesis and incorporation of spectrin into the cell membrane. In this system, EP promotes these effects without observable stimulation of progenitor proliferation in addition to that caused by the virus alone. Thus, the role of EP in terminal erythrocyte differentiation is not simply that of an erythroid-specific mitogen.
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Molecular cloning of biologically active proviral DNA of the anemia-inducing strain of spleen focus-forming virus.
Previously, we have molecularly cloned proviral DNA of a polycythemia-inducing strain of the spleen focus-forming virus (SFFVp). In this paper, we report that unintegrated proviral DNA of the anemia-inducing strain of SFFV (SFFVA) has been molecularly cloned into pBR322. This molecularly cloned DNA retains the biological activity of SFFVA, as infectious SFFV can be recovered from the DNA clone by marker rescue using a previously described two-stage cotransfection assay (Linemeyer et al., J. Virol. 35:710-721, 1980). The recovered SFFV retains an important property of the initial SFFVA which distinguishes SFFVA from SFFVP, namely, the ability of SFFVA to cause proliferation of erythroid cells in which hemoglobin synthesis is erythropoietin dependent. By utilizing a marker rescue technique, the splenomegaly and anemia characteristic of SFFVA-induced disease have been traced to a DNA fragment of SFFVA containing sequences coding for the env gene product. gp52. The results suggest that the differences in pathogenicity between SFFVP disease and SFFVA disease are an intrinsic property of the env gene products of these two variants of Friend virus, and future studies with the molecular clones of each strain should allow us to map regions of each env gene responsible for common and distinctive features of the erythroproliferative diseases induced by each virus.
Anemia↗