A reappraisal of the role of insulin-like growth factor I in the regulation of human hematopoiesis.

IGF-I has been reported to increase hematopoietic progenitor cell cloning efficiency. To investigate this phenomenon, we studied the IGF-I responsiveness of human marrow cells expressing IGF-I receptor (IGF-IR), a direct strategy not used previously. IGF-IR+ and control CD34+ marrow cells were isolated using immunoaffinity methods. Then, the cells were cloned in methylcellulose containing variable amounts of serum- and lineage-appropriate growth factors supplemented with recombinant human IGF-I. In contrast to CD34+ cells, IGF-IR+ cells never gave rise to CFU-Blast, CFU-Mix, CFU-GM, BFU-E, or CFU-E. To substantiate the suggestion that CD34+ and IGF-IR+ cells were distinct populations, we used reverse transcription PCR to detect IGF-I, EpO, and KIT receptor mRNAs in these cells. The mRNA phenotype of CD34+ cells was EpO (+), KIT (+), and IGF-IR (-), while IGF-IR+ cells were IGF-IR (+), EpO (-), and KIT (-). These results suggested that IGF-IR is either not expressed or expressed at low levels on normal hematopoietic progenitor cells. Functional significance of the latter possibility was tested by exposing CD34+ cells to IGF-IR antisense oligodeoxynucleotides. Colony formation was unaffected by oligodeoxynucleotide disruption of IGF-IR, suggesting that, even if expressed at low level, the receptor's functional significance was doubtful. Nevertheless, when cultured in the presence of IGF-I, IGF-IR+ cells elaborated an activity with mild BFU-E stimulatory effects. Accordingly, if IGF-I plays a role in hematopoietic colony formation, it is probably and results from stimulation of IGF-IR-positive ancillary cells to secrete growth factors. Studies carried out with human leukemia cells yielded similar results.

Evidence for a functional kit receptor in melanoma, breast, and lung carcinoma cells.

We sought to determine the functional significance of the c-kit receptor (Kit) in melanoma, breast carcinoma, and non-small cell lung cancer (NSCLC). To explore these issues, we first screened cell lines of each type for c-kit mRNA expression using a reverse-transcription polymerase chain reaction. We found that WM-39 melanoma cells, HTB-22 breast carcinoma cells, and A549 NSCLC cells all expressed c-kit mRNA. Of interest, all of these cells expressed the c-kit ligand, Steel factor (SF). We then assessed the functional significance of c-kit and SF expression by disrupting the gene's expression with antisense (AS) oligodeoxynucleotides (ODN) targeted to c-kit mRNA codons 1-6 and SF mRNA codons 2-7, respectively. Nonhybridizing sequences [sense (5) and scrambled (SCR)] were also employed as controls. WM-39, HTB-22, and A549 cells were exposed to ODN (approximately 25 microM) for 5-7 days. Downregulation of c-kit and SF mRNA, and c-kit protein was demonstrated in cells treated with AS ODN. Effects on viable cell growth were demonstrated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 -sulfophenyl)- 2H-tetrazolium (MTS) assay. In fact, c-kit antisense ODN inhibited the viable cell growth of A549 cells 66% and 79% compared to sense and untreated controls (P = .0003; P < .0001). Additionally, WM-39 cell growth was inhibited 48% and 21% (P < .0001, P < .03) and HTB-22 cell growth was inhibited 50% (P < .001) compared to sense and untreated controls. Viable cell growth was also significantly inhibited by SF AS ODN compared to S and SCR controls in all cell lines. These results demonstrate that WM-39, HTB-22, and A549 NSCLC cells all express the c-kit and SF protooncogenes and suggest that the encoded receptor and ligand are important for cell growth. By finding the presence, and functional importance, of both the receptor and ligand in these cells, this study suggests the existence of an autocrine loop growth mechanism worthy of further study.

Influence of different temperature of cryopreservation on the proliferative potential of human bone marrow progenitor cells. Transplantological implications.

The influence of two different temperatures of cryopreservation of human bone marrow on the proliferative potential of granulocyto-monocytic (GM-CFU), erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors was investigated. For this purpose, the human bone marrow cells were cryopreserved in the standard freezing medium supplemented with 10% DMSO and stored at -80 degrees C in a freezer or at -196 degrees C in a liquid nitrogen tank. Subsequently, cells were thawed, plated and stimulated in vitro to grow myeloid colonies. Unexpectedly, bone marrow cells stored at -80 degrees C formed about 60%, and those stored at -196 degrees C formed barely 8% of CFU-GM and BFU-E colonies respectively, in comparison to control non-frozen cells. CFU-Meg progenitors appeared to be the most sensitive to cryopreservation among all clonogeneic cells tested. The number of CFU-Meg derived colonies decreased to 20% in marrow cryopreserved at -80 degrees C and to about 2% in cells stored in liquid nitrogen at -196 degrees C. These data demonstrate the advantages of bone marrow storage at -80 degrees C over freezing in liquid nitrogen at -196 degrees C. Moreover, the increased sensitivity of CFU-Meg to cryopreservation could explain, at least partially, the clinical phenomenon of protracted thrombocytopenia observed in patients transplanted with the cryopreserved bone marrow cells.

The influence of 4 degrees C storage on proliferative potential of human bone marrow CD34+ cells. Transplantological implications.

The possibility of storage of human bone marrow CD34+ cells at 4 degrees C was investigated for bone marrow transplantational purposes. The cells were placed in Iscove medium supplemented with 20% serum (15% bovine calf serum 5% human AB serum) for three weeks at 4 degrees C. During the storage time, clonogenecity of granulocyto-monocytic (CFU-GM) erythropoietic (BFU-E) and megakaryocytic (CFU-Meg) progenitors was investigated. It was found that it is possible to keep human CD34+ cells at 4 degrees C, at least for a few days before transplantation. At day four of the storage, the number of CFU-GM and BFU-E cells still exceeded 50% of the cells present at day 0. However, we have found that CFU-Meg in comparison to other progenitors are much more sensitive to metabolic storage stress. This enhanced sensitivity of megakaryocytic progenitors could explain, at least partially, the well known phenomenon of retardation of the thrombopoietic recovery in patients undergoing bone marrow transplantations.

Cytokine stimulation of CD34+ bone marrow cells prior to cryopreservation enhances their post-thawing proliferative potential.

Marrow aplasia remains a significant cause of morbidity and mortality in the peri-transplant period. Administration of recombinant human hematopoietic growth factors along with the marrow graft is a widely used strategy to ameliorate this problem. Though arguably effective, this approach is extremely expensive. To develop alternative strategies for stimulating marrow engraftment, we investigated the utility of stimulating CD34+ enriched bone marrow cells with cytokines prior to cryopreservation. We found that culturing these cells for 1 to 3 days in Iscove's medium supplemented with kit ligand (KL), interleukin-3 (IL-3), interleukin-1 beta (IL-1 beta) and 20% bovine calf serum before freezing doubled the proliferative capacity of both myeloid and erythroid progenitor cells after thawing. Confirmation of increased proliferative activity in vivo would suggest that this approach might significantly shorten the period of marrow aplasia post transplant at far less expensive means.

[Storage of human bone marrow before transplantation at 4 degrees C. The effect of cell proliferation potential particularly in hematopoietic cloned cell lines]. / Przechowywanie ludzkiego szpiku przed przeszczepieniem w temperaturze 4 degrees C. Wplyw na potencjal proliferacyjny komórek poszczególnych linii krwiotwórczych.

The possibility of storage of human bone marrow CD34+ cells at 4 degrees C for bone marrow transplantation purposes was investigated. The cells were placed in Iscove medium supplemented with 20% serum (15% bovine calf serum + 5% human AB serum) for three weeks at 4 degrees C. During storage time clonogenicity of granulocytic-monocytic (CFU-GM) erythropoietic (BFU-E) and megakaryocytic (CFU-Meg) progenitors were investigated. It turns out that it is possible to keep human CD34+ cells at 4 degrees C for at least few days before transplantation. At day four of storage the number of CFU-GM and BFU-E cells still exceeded 50% of cells present at day 0. We have found however, CFU-Meg progenitors in comparison to others are much more sensitive to metabolic storage stress. This enhanced sensitivity of megakaryocytic progenitors could explain at least partially the well known ++phenomenon of retardation of thrombopoietic recovery in patients undergoing bone marrow transplantation.

STK-1 receptor gene is expressed in various human non-hematopoietic tumor cell lines--RT-PCR directed analysis of STK-1 mRNA expression. Preliminary report.

The aim of this study was to evaluate the presence of the mRNA for the hematopoietic STK-1 receptor in various human tumor cell lines using RT-PCR directed analysis of gene expression. We have found STK-1R mRNA to be expressed in non-hematopoietic malignant cells of ectodermal, endodermal and mesodermal origin confirms a hypothesis that STK-1R is similar to other receptors with intrinsic tyrosine kinase activity, is not for specific hematopoiesis and is also expressed in non-hematopoietic cells. The fact that STK-1R is detectable in different tumor cell lines should also inspire further studies to evaluate its role in the transformation and growth of malignant cells.

[Comparison of human granulocytopoiesis and monocytopoiesis stimulation in vitro with various growth factors: clinical conclusions]. / Porównanie stymulacji ludzkiej granulocytopoezy i monopoezy in vitro za pomoca rózych czynników wzrostu: wnioski kliniczne.

Different combination of human growth factors were checked for their ability to stimulate human granulocyte-monocytopoiesis in vitro. The optimal combination was found to be: Interleukin -3 (IL-3) + Granulocyte--Monocyte Colony Stimulating Factor (GM-CSF) + kit ligand (KL). In protocols concerning treatment of leukopenias with hemopoietic growth factors is should be concerned the use of combination of above listed growth factors. These growth factors could be added separately or in form of fusion proteins. In the future the efficiency of sequential addition of hemopoietic growth factors should be also evaluated.

[In-vitro study of anemia in chronic inflammatory conditions. Effect of recombined interleukin-1 beta on human erythropoiesis]. / Badania in vitro nad niedokrwistoscia towarzyszaca przewleklym stanom zapalnym. Wplyw remombinowanej interleukiny-1 beta na ludzka erytropoezee.

Study was inspired by the suggestion that interleukin 1-beta (IL-1 beta), one of the major inflammatory mediators, is directly responsible for anaemia of chronic inflammatory conditions. Effect of IL-beta was studied in vitro on methyl cellulose cultures of normal human bone marrow cells, the inoculate having been previously highly enriched in progenitor and stem cells. Our data did not support results reported by others that IL-1 beta is a strong inhibitor of early stages of erythropoiesis in vitro measured by early and late BFU-E formation. About 30% statistically not significant CFU-E inhibition was observed. Above studies show that IL-1 beta role in the development of anaemia associated with chronic inflammatory diseases requires further studies.

The storage of cells from different tumor lines in a mechanical freezer at -80 degrees C. Comparison to cryopreservation in liquid nitrogen.

This paper demonstrates that it is possible to store cells from different tumor lineages in a mechanical freezer at -80 degrees C. Tumor cells cryopreserved without controlled-rate freezing in medium containing 10% dimethyl sulfoxide (DMSO) and 10% Bovine Calf Serum (BCS) for 6 months displayed similar post-thawing viability as cells cryopreserved in liquid nitrogen at -196 degrees C. We recommend this method of cryopreservation as a convenient and less expensive alternative to liquid nitrogen storage. Further studies concerning the maximum storage time at -80 degrees C and studies comparing the optimal composition of the storage medium will further define the limitations and potential of this method.

Pre-stimulation of the human bone marrow CD34+ cells before storage at 4 degrees C with the early acting cytokines enhance their survival and increase proliferative potential. Transplantological implications.

The aim of this study was to evaluate whether short prestimulation of the human bone marrow CD34+ cells before storage at 4 degrees C with early acting cytokines would improve their subsequent survival and clonogenecity. To address this question the cells were placed in iscove medium supplemented with 20% BCS and stimulated for 24 hours in 37 degrees C incubator with different combination of the early acting cytokines (KL, IL-1 beta, IL-3, IL-6). Subsequently the cells were shifted to 4 degrees C. On day 6 the cells were plated in methylcelulose cultures and stimulated to growth CFU-GM colonies. It had been found that 24 hours long pre-stimulation with early acting cytokines significantly enhances the survival of the CD34+ clonogeneic progenitors stored subsequently at 4 degrees C. The CD34+ cell prestimulated for 24 hours at 37 degrees C with KL + IL-3 and than shifted to 4 degrees C formed more than twice as much CFU-GM colonies in comparison to the cells stored whole time at 4 degrees C even in the presence of KL + IL-3. Therefore we suggest, that if there is a need to store bone marrow cells before transplantation for short time they should be prestimulated with early acting cytokines for 24 hours in liquid culture at 37 degrees C first and than placed at 4 degrees C. The data presented in this paper should be in the future confirmed in in vivo SCID animal model to evaluate the repopulation ability of marrow cells pre-stimulated before short term storage at 4 degrees C with early acting cytokines to enhance the proliferative potential of the transplant and to shorten the dangerous aplastic phase in the patients after transplantation.

Expression and physiologic significance of Kit ligand and stem cell tyrosine kinase-1 receptor ligand in normal human CD34+, c-Kit+ marrow cells.

To determine the potential role of autocrine growth factor production in regulating primitive human hematopoietic cell development, we examined highly purified CD34+, c-Kit+ marrow mononuclear cells for expression of c-Kit ligand (KL) and stem cell tyrosine kinase 1 (stk1) ligand (STK1-L). Normal marrow mononuclear cells coexpressing CD34 and c-Kit were isolated by a combination of immunomagnetic bead isolation and fluorescence-activated cell sorting. Purified cells were then screened for expression of KL and stk1-L mRNA using a sensitive reverse transcription-polymerase chain reaction method. Using this approach, expression of both cytokine genes at the mRNA level was found in this highly enriched cell population. We then examined the functional significance of these mRNAs by inhibiting their expression with antisense (AS) oligodeoxynucleotides (ODN). In comparison to untreated or control ODN treated cells, inhibition of KL led to a 70% and 89% inhibition in burst-forming unit-erythroid (BFU-E) and colony-forming unit-Mix (CFU-Mix) colonies but had no significant effect on CFU-granulocyte-macrophage (CFU-GM) cloning efficiency. In contrast, inhibition of STK1-L alone had no effect on colony formation. However, when STK1-L AS ODN was combined with KL AS ODN, additive inhibition of CFU-GM and CFU-MIX but not of BFU-E colonies was observed. These findings, along with those of our previous studies showing inhibition of primitive hematopoietic cell growth with antisense ODN directed towards the stk1 receptor, suggest the possibility that both receptor/ligand axes regulate primitive hematopoietic cell growth via an autocrine growth loop.

A functional analysis of protooncogene Vav's role in adult human hematopoiesis.

The Vav protooncogene is expressed almost exclusively in hematopoietic cells, but its role in regulating adult human hematopoietic cell development remains uncertain. To analyze Vav function in adult blood cell formation, we used antisense (AS) oligodeoxynucleotides (ODN) to disrupt its expression in normal and malignant human hematopoietic cells. Bone marrow or peripheral blood mononuclear cells (MNC) were obtained from consenting normal donors and patients with acute or chronic myelogenous leukemia (AML and CML, respectively) and polycythemia vera (PV). Adherent and T-cell-depleted (A-T-) MNC or CD34+ MNC were exposed to unmodified sense, antisense, or scrambled sequence ODN corresponding to codons 2-7 of Vav's mRNA sequence. Cells were then assayed for Vav mRNA expression by reverse transcription-polymerase chain reaction and Vav protein expression by Western binding. After showing that Vav-targeted AS ODN could specifically diminish Vav mRNA and protein expression, we assessed the ability of Vav-deficient cells to form myeloid and erythroid colonies in methyl-cellulose cultures. When normal CD34+ MNC were exposed to Vav AS ODN, no effect on colony-forming unit-granulocyte-macrophage (CFU-GM) or CFU-megakaryocyte colony formation was observed. In contrast erythroid colony growth was inhibited by a mean +/- SD of 62% +/- 16%. In patients with hematopoietic malignancies. Vav-targeted AS ODN inhibited CFU-GM colony formation in a sequence-specific and dose-dependent manner in 1 of 3 AML, 13 of 17 CML, and 2 of 2 PV patients. At the highest concentration used, the Vav AS ODN inhibited CFU-GM colony formation from 66% to 81% when compared with control cell colony growth. Burst-forming unit-erythroid (BFU-E) colony-formation was also assessed in 7 PV patients. The Vav-targeted AS ODN inhibited BFU-E colony formation in all by a mean +/- SD of 81% +/- 4%. These findings suggest that Vav function may not be easily complemented in a significant subset of normal adult erythroid progenitor cells and may also be necessary for myeloid progenitor cell growth in a variety of hematopoietic malignancies.