Normal Bone Marrow Progenitor Cells Research Articles

2′,2′‐Difluorodeoxycytidine (Gemcitabine) Induces Apoptosis in Myeloma Cell Lines Resistant to Steroids and 2‐Chlorodeoxyadenosine (2‐CdA)

The paucity of effective cytotoxic agents for the treatment of steroid resistant multiple myeloma explains the ongoing search for alternative substances for chemotherapy of this disease. In the present study, the purine antagonist 2-chlorodeoxyadenosine (2-CdA, cladribine) and the pyrimidine antagonist 2',2'-difluorodeoxycytidine (gemcitabine) were tested on four myeloma cell lines (i.e., U 266, OPM 2, RPMI 8226, IM 9), one plasma cell leukemia cell line (HS Sultan) and a myeloid control cell line (HL 60), all of which are resistant to 10-6 M dexamethasone. Gemcitabine has been found to be promising in the chemotherapy of other tumors with low proliferative activity, but its effectiveness against myeloma cells has not been analyzed so far. In our tests, gemcitabine induced a significant degree of apoptosis in all cell lines investigated. After incubation for 48 h with 10 microM gemcitabine, the median numbers of apoptotic cells were in the range of 45% in the OPM 2 and 79% in the U 266 cell line. All of the investigated cell lines were responsive to concentrations of 10 microM gemcitabine even after an exposure of only 30 min, three of them (U 266, HS Sultan, IM 9) also responded to a concentration of 10 nM. Higher concentrations and longer exposure times were necessary to suppress the growth of normal hematopoietic bone marrow progenitor cells. In contrast to gemcitabine, standard concentrations of 2-CdA (i.e., 30 and 300 nM) failed to induce a significant degree of apoptosis in the cell lines investigated but inhibited the growth of myeloid progenitor cells. The results suggest that gemcitabine induces apoptosis in myeloma and plasma cell leukemia lines resistant to steroids and 2-CdA. The fact that tumor cell apoptosis was achieved at concentrations clinically achievable and tolerable, which at the same time do not inhibit the growth of normal CFU-GM progenitor cells, favors the initiation of phase I trials with this drug for the treatment of multiple myeloma.

1118 Amifostine improves the antileukemic therapeutic index of mafosfamide: Implications for bone marrow purging

To test the potential use of amifostine (Ami) to protect normal bone marrow (N1 BM) progenitor cells during ex vivo purging of leukemia, the dose response of mafosfamide (Mfs) ± Ami on N1 BM progenitor cells was determined and the LD95 was calculated. Ami pretreatment resulted in a statistically significant protection of CFU-GM and erythroid blast-forming units (BFU-E) from Mfs toxicity. The mean ± SEM μg/ml LD95 concentrations were: CFU-GM, Mfs alone 54 ± 13 vs Ami-Mfs 66 ± 14, p. 005, BFU-E, Mfs alone 48 ± 14, vs Ami-Mfs 61 ± 11, p .005. In contrast, Ami pretreatment sensitized CFU-L to Mfs toxicity: LD95 for Mfs alone 33 ± 4 vs Ami-Mfs 27 ± 3, p .003. At the LD95 fur CFU-GM and BFU-E, Mfs alone resulted in a ≈4 log cell-kill for L-CFU. Ami protection of N1 BM resulting in a higher Mfs LD95 concentration, coupled with the Ami-sensitization of the leukemia, provided an estimated additional 6+ log leukemia cell-kill. This enhanced selective leukemia cell-kill from Mfs during ex vivo purging using the LD95 concentration of Mfs for normal marrow progenitors offers the potential for lowering the incidence of leukemic relapse while preserving a greater number of normal progenitor cells for engraftment following autologous transplantation.

Amifostine Improves the Antileukemic Therapeutic Index of Mafosfamide: Implications for Bone Marrow Purging

One of the principal challenges of cancer chemotherapy is the relative inability of most anticancer drugs to distinguish between normal and neoplastic tissues. Consequently, a broad range of toxicities are experienced by patients, especially myelosuppression. Amifostine, a phosphorylated aminothiol, increases the selectivity of specific anticancer drugs for neoplastic cells by protecting normal tissues. One potential application of this protector is during bone marrow purging to selectively remove contaminating cancer cells. This study took normal or leukemic marrow from human subjects and evaluated the ability of amifostine to selectively protect normal bone marrow progenitor cells versus leukemic progenitor cells from the cytotoxic effect of mafosfamide. The dose response of mafosfamide amifostine on leukemia colony-forming units or normal marrow progenitor cells was determined and the LD95 was calculated. Amifostine pretreatment resulted in a statistically significant protection of granulocyte-macrophage colony-forming units and erythroid blast-forming units from the toxicity of mafosfamide (P = .031). Thus, amifostine protection of normal marrow progenitor cells allows a higher LD95 concentration of mafosfamide to be used in ex vivo purging. In contrast, amifostine pretreatment increased the cytotoxicity of mafosfamide on the fresh human leukemia progenitor cells (P = .006). The dual effect of amifostine protection of normal marrow progenitor cells coupled with amifostine-induced sensitization of the leukemia cells increases the possible cell-kill of leukemic stem cells. With amifostine pretreatment, at the LD95 concentrations of mafosfamide for marrow progenitor cells, there was an estimated 6 log increase in cell-kill of the leukemia cells. This selective cell-kill offers the potential for lowering the incidence of leukemic relapse, while preserving more normal stem cells for autologous transplantation.

Amifostine improves the antileukemic therapeutic index of mafosfamide: implications for bone marrow purging

One of the principal challenges of cancer chemotherapy is the relative inability of most anticancer drugs to distinguish between normal and neoplastic tissues. Consequently, a broad range of toxicities are experienced by patients, especially myelosuppression. Amifostine, a phosphorylated aminothiol, increases the selectivity of specific anticancer drugs for neoplastic cells by protecting normal tissues. One potential application of this protector is during bone marrow purging to selectively remove contaminating cancer cells. This study took normal or leukemic marrow from human subjects and evaluated the ability of amifostine to selectively protect normal bone marrow progenitor cells versus leukemic progenitor cells from the cytotoxic effect of mafosfamide. The dose response of mafosfamide amifostine on leukemia colony-forming units or normal marrow progenitor cells was determined and the LD95 was calculated. Amifostine pretreatment resulted in a statistically significant protection of granulocyte-macrophage colony-forming units and erythroid blast-forming units from the toxicity of mafosfamide (P = .031). Thus, amifostine protection of normal marrow progenitor cells allows a higher LD95 concentration of mafosfamide to be used in ex vivo purging. In contrast, amifostine pretreatment increased the cytotoxicity of mafosfamide on the fresh human leukemia progenitor cells (P = .006). The dual effect of amifostine protection of normal marrow progenitor cells coupled with amifostine-induced sensitization of the leukemia cells increases the possible cell-kill of leukemic stem cells. With amifostine pretreatment, at the LD95 concentrations of mafosfamide for marrow progenitor cells, there was an estimated 6 log increase in cell-kill of the leukemia cells. This selective cell-kill offers the potential for lowering the incidence of leukemic relapse, while preserving more normal stem cells for autologous transplantation.

Conditioned Media from a Cell Strain Derived from a Patient with Mastocytosis Induces Preferential Development of Cells That Possess High Affinity IgE Receptors and the Granule Protease Phenotype of Mature Cutaneous Mast Cells

We have demonstrated for the first time that a conditioned medium from a human cell strain can induce morphologically mature mast cells that express Fc epsilon RI and three mast cell-specific proteases from normal bone marrow progenitor cells. In contrast, recombinant human Kit ligand induced the differentiation of mast cells that were tryptase-positive but negative for chymase, carboxypeptidase, and Fc epsilon RI. This data indicates that factors other than Kit ligand are critical for inducing the differentiation and maturation of mast cells in the human. The HBM-M cell was originally derived from a patient with mastocytosis. As mastocytosis is thought to represent a reactive hyperplasia rather than a mast cell malignancy, the factor secreted by the HBM-M cell strain could well be responsible for the mast cell hyperplasia seen in some patients with mastocytosis.

Distinct and overlapping direct effects of macrophage inflammatory protein-1 alpha and transforming growth factor beta on hematopoietic progenitor/stem cell growth

Both transforming growth factor beta (TGF beta) and macrophage inflammatory protein 1 alpha (MIP-1 alpha) have been shown to be multifunctional regulators of hematopoiesis that can either inhibit or enhance the growth of hematopoietic progenitor cells (HPC). We report here the spectrum of activities of these two cytokines on different hematopoietic progenitor and stem cell populations, and whether these effects are direct or indirect. MIP-1 alpha enhances interleukin-3 (IL- 3)/and granulocyte-macrophage colony-stimulating factor (GM- CSF)/induced colony formation of normal bone marrow progenitor cells (BMC) and lineage-negative (Lin-) progenitors, but has no effect on G- CSF or CSF-1/induced colony formation. Similarly, TGF beta enhances GM- CSF/induced colony formation of normal BMC and Lin- progenitors. In contrast, TGF beta inhibits IL-3/ and CSF-1/induced colony formation of Lin- progenitors. The effects of MIP-1 alpha and TGF beta on the growth of Lin- progenitors were direct and correlate with colony formation in soft agar. Separation of the Lin- cells into Thy-1 and Thy-1lo subsets showed that the growth of Thy-1lo Lin- cells is directly inhibited by MIP-1 alpha and TGF beta regardless of the cytokine used to stimulate growth (IL-3), GM-CSF, or CSF-1). In contrast, two other stem cell populations (0% to 15% Hoechst 33342/Rhodamine 123 [Ho/Rh123] and Lin- Sca-1+ cells) were markedly inhibited by TGF beta and unaffected by MIP- 1 alpha. Furthermore, MIP-1 alpha has no effect on high proliferative potential colony-forming cells 1 or 2 (HPP-CFC/1 or /2) colony formation in vitro, whereas TGF beta inhibits both HPP-CFC/1 and HPP- CFC/2. Thus, MIP-1 alpha and TGF beta are direct bidirectional regulators of HPC growth, whose effects are dependent on other growth factors present as well as the maturational state of the HPC assayed. The spectrum of their inhibitory and enhancing activities shows overlapping yet distinct effects.

Distinct and Overlapping Direct Effects of Macrophage Inflammatory Protein-la and Transforming Growth Factor β on Hematopoietic Progenitor/Stem Cell Growth

Both transforming growth factor beta (TGF beta) and macrophage inflammatory protein 1 alpha (MIP-1 alpha) have been shown to be multifunctional regulators of hematopoiesis that can either inhibit or enhance the growth of hematopoietic progenitor cells (HPC). We report here the spectrum of activities of these two cytokines on different hematopoietic progenitor and stem cell populations, and whether these effects are direct or indirect. MIP-1 alpha enhances interleukin-3 (IL-3)/and granulocyte-macrophage colony-stimulating factor (GM-CSF)/induced colony formation of normal bone marrow progenitor cells (BMC) and lineage-negative (Lin-) progenitors, but has no effect on G-CSF or CSF-1/induced colony formation. Similarly, TGF beta enhances GM-CSF/induced colony formation of normal BMC and Lin- progenitors. In contrast, TGF beta inhibits IL-3/ and CSF-1/induced colony formation of Lin- progenitors. The effects of MIP-1 alpha and TGF beta on the growth of Lin- progenitors were direct and correlate with colony formation in soft agar. Separation of the Lin- cells into Thy-1 and Thy-1lo subsets showed that the growth of Thy-1lo Lin- cells is directly inhibited by MIP-1 alpha and TGF beta regardless of the cytokine used to stimulate growth (IL-3), GM-CSF, or CSF-1). In contrast, two other stem cell populations (0% to 15% Höechst 33342/Rhodamine 123 [Hö/Rh123] and Lin-Sca-1+ cells) were markedly inhibited by TGF beta and unaffected by MIP-1 alpha. Furthermore, MIP-1 alpha has no effect on high proliferative potential colony-forming cells 1 or 2 (HPP-CFC/1 or /2) colony formation in vitro, whereas TGF beta inhibits both HPP-CFC/1 and HPP-CFC/2. Thus, MIP-1 alpha and TGF beta are direct bidirectional regulators of HPC growth, whose effects are dependent on other growth factors present as well as the maturational state of the HPC assayed. The spectrum of their inhibitory and enhancing activities shows overlapping yet distinct effects.

Comments on "Radiation Dose-Fractionation and Dose-Rate Relationships for Long-Term Repopulating Hemopoietic Stem Cells in a Murine Bone Marrow Transplant Model" by R. van Os, H. Thames, A. W. T. Konings and J. D. Down (Radiat. Res. 136, 118-125, 1993)

We have read with great interest the paper by van Os et al. regarding the dose-fractionation and dose-rate relationships for the sterilization of bone marrow stem cells by totalbody irradiation (TBI) (1). Using an ingenious syngeneic murine chimera model, the authors unequivocally demonstrated that fractionated or single-dose TBI at a low dose rate was less effective than single-dose TBI at a high dose rate in depleting subsets of bone marrow stem cells in mice. This observation is consistent with the belief that bone marrow stem cells are capable of repairing sublethal radiation damage, but it is at variance with the general notion that the hemopoietic stem cells are characteristic of limited radiation repair capacity. While we are in total agreement with Dr. van Os and his colleagues regarding the radiosensitivity of bone marrow stem cells, we would like to note that we reported essentially the same conclusion more than 10 years ago. Based on our LD50/30 study using a murine model (2), and also on numerous experimental data reported by other investigators on the response of bone marrow stem cells to single-dose irradiation at different dose rates or to fractionated irradiations, we concluded that bone marrow stem cells possess a considerable capacity to repair sublethal radiation damage (3, 4). Our conclusion was based mostly on studies on the radiation repair capacity of bone marrow progenitor cells in vitro. Note that in vitro culture conditions are likely to result in an underestimation of the repair capacity of normal bone marrow progenitor cells. In recent studies, we reported that several cytokines, which are found at high concentrations within the bone marrow microenvironment, are able to enhance the ability of normal hemopoietic progenitor cell subpopulations CFU-GEMM, CFU-GM, CFU-E and BFU-E to repair sublethal radiation damage (5). Indications are that the radiation response of leukemic progenitor cells is not different from that of normal bone marrow progenitor cells. Fractionated or low-dose-rate TBI may allow leukemic progenitor cells to repair sublethal radiation damage. We have demonstrated that HL-60 cells, an established human acute promyelocytic eukemic cell line (6), as well as many of the primary clonogenic blasts from acute lymphoblastic leukemia patients (7), indeed possess a considerable capacity to repair sublethal radiation damage, and some of these are extremely radioresist nt (8). In this connection, we would like to point out that t e long-term survival among high-risk acute lymphoblastic leukemia (ALL) patients who receive bone marrow transplantation after TBI is only 5-15% because of recurrence of the leukemia. We also found that the number of residual

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate down-regulates expression of the c-kit proto-oncogene product.

Modulation of expression of the c-kit proto-oncogene product, the receptor for the recently identified stem cell factor, was studied on 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated cultures of CD34+ normal bone marrow progenitor cells, blast cells from patients with primary acute myelogenous leukemia, cells from the leukemia cell lines HEL and MO7E, as well as cultured HMC-1 mast cells. Expression of c-kit was assessed on both RNA and protein level employing standard Northern blotting, reverse transcription, and polymerase chain reaction-based Southern blotting, as well as cell surface labeling with anti-c-kit mAb YB5.B8. Treatment of virtually all cell types with nontoxic concentrations of TPA (10(-9) M) for at least 48 h was associated with down-regulation of synthesis of c-kit transcripts and stem cell factor-receptor surface expression. Studies on the mechanism of action of TPA utilizing the HEL erythroleukemia line showed that TPA was primarily acting by accelerating the turnover of c-kit RNA most likely through induction of a destabilizing protein. The effect of TPA on c-kit expression levels was independent of TPA-mediated induction of differentiation since other compounds including IFN-gamma, vitamin D3, retinoic acid, arabinofuranosylcytosine, butyric acid, and camptothecin, which also effectively induced differentiation of HEL cells, failed to alter levels of c-kit expression.

Anti-human immunodeficiency virus type 1 activity of an anti-CD4 immunoconjugate containing pokeweed antiviral protein.

The ability of an alpha CD4-pokeweed antiviral protein (PAP) immunoconjugate to inhibit replication of human immunodeficiency virus type 1 (HIV-1) was evaluated in vitro with 22 clinical HIV-1 strains obtained from four seropositive asymptomatic individuals, three patients with AIDS-related complex, and four patients with AIDS. Fifteen isolates were from zidovudine-untreated individuals, whereas seven isolates were obtained after 24 to 104 weeks of therapy with zidovudine, alone or alternating with zalcitabine. Mean zidovudine 50% inhibitory concentrations (IC50s) were 126 nM (range, 1 to 607 nM) for isolates from zidovudine-untreated individuals and 2,498 nM (range, 14 to 6,497 nM) for strains from patients treated with antiretroviral agents. Mean alpha CD4-PAP IC50s were 48 x 10(-3) nM (range, 0.02 x 10(-3) to 212 x 10(-3) nM) for isolates from zidovudine-untreated individuals, and 16 x 10(-3) nM (range, 2 x 10(-3) to 28 x 10(-3) nM) for isolates from treated patients. Overall, higher concentrations of alpha CD4-PAP were necessary to inhibit HIV-1 strains from untreated individuals at more advanced stages of disease. Seventeen isolates were susceptible to zidovudine (mean IC50, 117 nM), and five were resistant to zidovudine (mean IC50, 3,724 nM). Mean alpha CD4-PAP IC50s were 43 x 10(-3) nM for zidovudine-susceptible isolates and 19 x 10(-3) nM for isolates resistant to zidovudine. All HIV-1 strains had IC50s greater than 0.5 nM for unconjugated PAP, the alpha CD19-PAP immunoconjugate, and monoclonal antibody alpha CD4. At concentrations as high as 5,000 nM, alphaCD4-PAP did not inhibit colony formation by normal bone marrow progenitor cells(BFU-E, CFU-GM , and CFU-GEMM) or myeloid cell lines (KG-1 and HL-60) and did not decrease cell viabilities of T-cell (Jurkat) or B-cell (FL-112 and Raji) precursor lines. Overall, alphaCD4-PAP demonstrated more potent anti-HIV-1 activity than zidovudine and inhibited replication of zidovudine-susceptible and zidovudine-resistant viruses at concentrations that were not toxic to lymphohematopoietic cell populations.

A Bispecific Antibody Enhances Cytokine-Induced Killer-Mediated Cytolysis of Autologous Acute Myeloid Leukemia Cells

An anti-CD3 Fab' x anti-CD13 Fab' bispecific antibody (BsAb) was generated. This BsAb reacted with both CD3+ T cells and CD13+ acute myeloid leukemia (AML) cells. We investigated whether cytokine-stimulated peripheral blood mononuclear cells (PBMC) could lyse patient AML cells after addition of the BsAb. When interleukin-2 (IL-2)-stimulated PBMC were assayed for their cytotoxicity against 51Cr-labeled allogeneic and autologous CD13+ AML cells, their activity was markedly enhanced by the addition of the BsAb. PBMC stimulated with IL-2 plus anti-CD3 monoclonal antibody (MoAb) showed higher proliferative ability and higher cytotoxicity if this was expressed as lytic units per culture. IL-7-stimulated PBMC also exhibited enhanced cytotoxicity against CD13+ AML cells after addition of the BsAb. Ultrastructurally, CD13+ AML cells incubated with IL-2 plus anti-CD3 MoAb-stimulated PBMC and the BsAb showed apoptotic morphologic changes. A colony assay for AML blast progenitors showed that the colony formation of CD13+ AML cells was inhibited by the addition of autologous IL-2 plus anti-CD3 MoAb-stimulated PBMC, and that this inhibition was further enhanced by the addition of the BsAb. A colony assay for normal bone marrow progenitor cells showed that the addition of autologous IL-2 plus anti-CD3 MoAb-stimulated PBMC and the BsAb inhibited the formation of granulocyte-macrophage colonies and mixed-cell colonies. However, the degree of inhibition was smaller than that for the AML blast colonies. Taken together, these findings suggest that this BsAb may be useful for ex vivo purging of CD13+ AML cells in autologous bone marrow transplantation.

A bispecific antibody enhances cytokine-induced killer-mediated cytolysis of autologous acute myeloid leukemia cells

An anti-CD3 Fab' x anti-CD13 Fab' bispecific antibody (BsAb) was generated. This BsAb reacted with both CD3+ T cells and CD13+ acute myeloid leukemia (AML) cells. We investigated whether cytokine- stimulated peripheral blood mononuclear cells (PBMC) could lyse patient AML cells after addition of the BsAb. When interleukin-2 (IL-2)- stimulated PBMC were assayed for their cytotoxicity against 51Cr- labeled allogeneic and autologous CD13+ AML cells, their activity was markedly enhanced by the addition of the BsAb. PBMC stimulated with IL- 2 plus anti-CD3 monoclonal antibody (MoAb) showed higher proliferative ability and higher cytotoxicity if this was expressed as lytic units per culture. IL-7-stimulated PBMC also exhibited enhanced cytotoxicity against CD13+ AML cells after addition of the BsAb. Ultrastructurally, CD13+ AML cells incubated with IL-2 plus anti-CD3 MoAb-stimulated PBMC and the BsAb showed apoptotic morphologic changes. A colony assay for AML blast progenitors showed that the colony formation of CD13+ AML cells was inhibited by the addition of autologous IL-2 plus anti-CD3 MoAb-stimulated PBMC, and that this inhibition was further enhanced by the addition of the BsAb. A colony assay for normal bone marrow progenitor cells showed that the addition of autologous IL-2 plus anti- CD3 MoAb-stimulated PBMC and the BsAb inhibited the formation of granulocyte-macrophage colonies and mixed-cell colonies. However, the degree of inhibition was smaller than that for the AML blast colonies. Taken together, these findings suggest that this BsAb may be useful for ex vivo purging of CD13+ AML cells in autologous bone marrow transplantation.

Expression of the human hematopoietic progenitor cell antigen CD34 in vascular and spindle cell tumors

The human hematopoietic progenitor cell antigen is known as CD34. This antigen is present on normal bone marrow progenitor cells and vascular endothelial cells. We used the monoclonal antibody anti-CD34 and immunoperoxidase staining techniques to evaluate the expression of CD34 in benign and malignant vascular and spindle cell tumors. All of the 42 vascular lesions, except two of three lesions of intravascular papillary endothelial hyperplasia, demonstrated diffuse membraneous staining of moderate to strong intensity of their endothelial cells. Also, normal placentas (five) showed similar staining. All neurofibromas (12), three of five neuromas, and one of four neurilemmomas revealed moderate to strong, diffuse, membraneous staining. Five of eight piloleiomyomas, two of seven angioleiomyomas, and one of five uterine leiomyomas showed focal to diffuse, and weak to moderate, membraneous staining in the smooth muscle component. Six dermatofibrosarcoma protuberans were studied: generalized, strongly positive membraneous staining was present in four. All specimens showed staining of the normal endothelial cells and the cells surrounding the hair follicles (bulge area), sebaceous glands, and eccrine glands. No staining was demonstrated in any of the following fibrohistiocytic tumors: atypical fibroxanthomas (two), fibrous dermatofibromas (23), giant cell tumor of tendon sheath (one), and hemosiderotic dermatofibromas (18). Melanocytic tumors [Spitz nevi (three) and spindle cell superficial spreading malignant melanoma (one)], Merkel cell carcinomas (six), and spindle cell squamous cell carcinomas (two) did not stain with anti-CD34. Glomus tumors (two) and a hemangiopericytoma were also negative except for their vascular channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Recombinant Interleukin‐3 and Recombinant Interleukin‐6 on Radiation Survival of Normal Human Bone Marrow Progenitor Cells

AbstractThe effects of recombinant interleukin‐6 (rIL‐6) and rIL‐3 on the radiation responses of myeloid [colony forming unit‐granulocyte‐macrophage (CFU‐GM)] and erythroid [burst forming unit‐erythroid (BFU‐E)] human pediatric bone marrow progenitor cells were analyzed. Cytokine exposure times of 30 min, 1 hr, 2 hr, 4 hr, 16 hr, and 24 hr were used prior to irradiation. The most notable observations were based on short exposure data, which provided unprecedented evidence that rIL‐6 increases the radiation damage repair capacity of both CFU‐GM and BFU‐E without affecting their radiation sensitivity, whereas rIL‐3 modulated the radiation responses of CFU‐GM only. The cytokine‐induced increase in radiation survival of CFU‐GM or BFU‐E at doses ≤200 cGy appeared to be primarily due to an increase in the ability of these hematopoietic progenitor cell populations to repair sublethal radiation damage.

Retroviral mediated expression of CD18 in normal and deficient human bone marrow progenitor cells.

The CD18 gene encodes the beta subunit of leukocyte integrins, and autosomal recessive deficiency of CD18 in humans causes a life-threatening abnormality of granulocyte migration. A high titer amphotropic retrovirus encoding CD18 was used to infect bone marrow cells from normal and CD18-deficient human donors. Infected cells were maintained in a long-term culture system and analyzed (1) using PCR to detect the provirus in granulocyte or macrophage colonies (CFU-GMs) derived from the culture, (2) using reverse transcription-PCR (RT-PCR) to detect transcripts in the floating cells in the culture, and (3) using immunostaining of the floating cells to analyze expression of human CD18. By both cocultivation and supernatant infection, a significant fraction (10-82%) of the CFU-GMs were positive for the provirus after five weeks in long-term culture, and as high as 10-15% of the floating cells were positive by immunostaining after nine weeks in the long-term culture. In all cases, cocultivation showed higher infection efficiency than supernatant infection. This is the first report of the introduction of human CD18 cDNA into the bone marrow progenitor cells of patients with leukocyte adhesion deficiency.

Granulocyte-Macrophage Colony-Stimulating Factor/Interleukin-3 Fusion Protein (pIXY 321) Enhances High-Dose Ara-C–Induced Programmed Cell Death or Apoptosis in Human Myeloid Leukemia Cells

AbstractHigh dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.

Granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein (pIXY 321) enhances high-dose Ara-C-induced programmed cell death or apoptosis in human myeloid leukemia cells

High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.

Comparison of cytotoxicity of the (−)- and (+)- enantiomer of 2′,3′- dideoxy-3′-thiacytidine in normal human bone marrow progenitor cells

The effects of racemic cis-2',3'-dideoxy-3'-thiacytidine [(±)-BCH-189] and its two enantiomers on human myeloid and erythroid colony-forming cells were studied by clonogenic assays. The (+)-isomer was the most toxic with a median inhibitory concentration approximating 2 μM in both cell lineages. In contrast, concentrations of the (−)-isomer required for 50% inhibition of granulocyte macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) were 33.9 ± 15.1 and 169.4 ± 87.9 μM, respectively. The racemic BCH-189 was quite toxic to these cells, but to a lesser extent than observed with 3′-azido-3′- deoxythymidine and 3′-fluoro-3′-deoxythymidine (positive controls).

Detection of surface antigen expression of pre- and post-cultured normal adult's bone marrow cells by using ABC technique and its significance.

The expression of surface antigens on both normal adult's bone marrow and multipotential progenitor cells (CFU-Mix) was detected by use of ABC technique. The culture system of CFU-Mix was an ideal model for studying the differentiation direction of hematopoietic cells. The monoclonal antibodies (McAb) CD3, CD4, CD8 specific for T-lymphocytic lineage, CD22 for B-lymphocytic lineage and CD11, CD13, CD14 for granulocytomonocytic lineage were all demonstrated in certain proportion in cultured CFU-Mix, which might be associated with the control of hematopoiesis. A decrease in the positive rate of OKT9 and CD34 in cultured cells might be related to the differentiation of hematopoietic cells. SZ-2 and SZ-21 could contribute to the identification of the presence of megakaryocytes in CFU-Mix.

Induction of endonuclease-mediated apoptosis in tumor cells by C-nitroso-substituted ligands of poly(ADP-ribose) polymerase.

6-Nitroso-1,2-benzopyrone and 3-nitrosobenzamide, two C-nitroso compounds that inactivate the eukaryotic nuclear protein poly(ADP-ribose) polymerase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, ADPRT, EC 2.4.2.30] at one zinc-finger site, completely suppressed the proliferation of leukemic and other malignant human cells and subsequently produced cell death. Tumoricidal concentrations of the drugs were relatively harmless to normal bone marrow progenitor cells and to superoxide formation by neutrophil granulocytes. The cellular mechanism elicited by the C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesium-dependent endonuclease. This endonuclease is maintained in a latent form by poly(ADP-ribosyl)ation, but inactivation of ADPRT by C-nitroso drugs derepresses the DNA-degrading activity. ADPRT is thus identified as a critical regulatory enzyme component of a DNA-binding multiprotein system that plays a central function in defining DNA structures in the intact cell.