Effect of platelet-derived growth factor on the proliferation of blast progenitors from acute myeloblastic leukemia patients

The effect of platelet-derived growth factor (PDGF) on self-renewal and terminal divisions of blast progenitors of acute myeloblastic leukemia was studied. The terminal divisions of blast progenitors were determined as the primary blast colony formation in methylcellulose culture; self-renewal was assessed by secondary colony formation by replating in methylcellulose and the recovery of clonogenic cells in suspension culture. PDGF neither enhanced nor suppressed primary or secondary blast colony formation in methylcellulose culture nor recovery of clonogenic cells in suspension. The results show that PDGF does not affect the self-renewal or terminal divisions of leukemic blast progenitors.     

Self-renewal in culture of proliferative blast progenitor cells in acute myeloblastic leukemia

We have proposed that colonies of cells with blastlike morphology growing in culture are derived from a blast subpopulation with high proliferative potential. To test whether or not these blast progenitors have the capacity for self-renewal, blast colonies grown from the peripheral blood of the 21 patients with acute myeloblastic leukemia were replated; secondary colonies were observed in 17 instances, and these were similar to primary colonies in size, morphology, and culture requirements. Great patient-to-patient variation was observed in the frequency of secondary colonies, but low secondary plating efficiency was significantly correlated with successful remission induction. We conclude that the blast progenitors detected in the assay have at least limited self-renewal capacity and that this capacity may, along with other risk factors, contribute to clinical outcome.     

Effect of interferon on pluripotent hemopoietic progenitors (CFU-GEMM) derived from human bone marrow

A culture assay for multilineage hemopoietic progenitors (CFU-GEMM) that form mixed hematopoietic colonies containing granulocytes, erythroblasts, megakaryocytes and macrophages was described. The effect of human leukocyte interferon on multilineage hemopoietic progenitors was examined. Leukocyte interferon does reduce the plating efficiency of noncommitted precursors (CFU-GEMM) in a dose related fashion. A reduction of the plating efficiency of mixed hemopoietic colonies was observed even when interferon was added 72 hours after initiation of the cultures.     

The in vitro growth patterns and drug sensitivities of leukemic blast progenitors among the subtypes of acute myelocytic leukemia.

The in vitro growth activities and drug sensitivities of leukemic blast progenitors were compared among the subgroups of acute myelocytic leukemia (AML) classified according to the French-American-British (FAB) cooperative group. Leukemic cells separated from the peripheral bloods of AML patients were cultured in methylcellulose media, and the plating efficiencies of primary colonies (PE1) and secondary colonies after replating (PE2) were determined. PE1 and PE2 have been considered to reflect the capacities of terminal divisions and self-renewal of leukemic blast progenitors, respectively. PE1 and PE2 were variable among AML patients; these findings suggest that AML is a heterogeneous disease in terms of the proliferative activities of leukemic cells. No significant correlation was noted between PE1 or PE2 and the AML subtype. The sensitivities to cytosine arabinoside (Ara-C) of leukemic blast progenitors were studied in methylcellulose and suspension cultures. Ara-C sensitivity was not significantly correlated with the AML subtype, either. In contrast, there was statistically significant correlation between PE2 and the remission outcome of the patients, whereas PE1 was not significantly associated with the clinical outcome. The results in the present study indicate that the proliferative activity, especially self-renewal capacity, of leukemic blast progenitors is highly predictive of the prognosis of AML patients but is not significantly correlated with the AML subtype classified by the blast morphology.     

Effects of purified human native granulocyte colony-stimulating factor (G-CSF) on proliferation of blast progenitors in acute myeloblastic leukemia

Blast progenitors in acute myeloblastic leukemia (AML) grow in methylcellulose and suspension cultures. Blast colony formation in methylcellulose culture reflects the terminal divisions of blast progenitors, while secondary colony formation, by replating in methylcellulose and recovering clonogenic cells in suspension culture, reflects the self-renewal of blast progenitors. To analyze the regulatory mechanisms of the proliferation of leukemic blast progenitors, the effects of highly purified native granulocyte colony-stimulating factor (G-CSF) obtained from human squamous cell carcinoma line (CHU-2) on blast progenitors in AML patients were studied in methylcellulose and suspension cultures. Purified G-CSF stimulated the growth of blast progenitors in both culture systems, although sensitivity to G-CSF varied from patient to patient. No obvious maturation of leukemic blasts was noted in suspension culture in the presence of G-CSF. The data suggest that a normal hemopoietic regulator may play a role in the growth of blast progenitors in AML patients.     

Comparison of in vitro growth characteristics of blast cell progenitors (CFU-L) in patients with myelodysplastic syndromes and acute myeloid leukemia.

Current knowledge is inadequate to explain the different patterns of blast cell accumulation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). We compared the growth patterns of blast cell progenitors (CFU-L) in 23 patients with advanced MDS and 32 patients with de novo AML. Circulating blast progenitors were identified in 74% of MDS and 81% of AML samples. Primary plating efficiencies (PE1) were similar in both disorders, despite marked differences in peripheral blast cell concentrations. By cytological and cytochemical examination, colonies from MDS patients were indistinguishable from those obtained in AML. Cell cycle status was assessed by loss of colony formation following short-term exposure to cytosine arabinoside. CFU-L suicide rates (median, range) were 40% (12% to 77%) in MDS and 60.5% (27% to 98%) in AML. Actively proliferating blast cell progenitors are thus not confined to AML, but are also present in the majority of MDS patients. An important difference between MDS and AML was found when self-renewal capacity of CFU-L was examined by means of secondary plating efficiencies (PE2). Colonies could be successfully replated in 74% of AML cases. PE2 showed marked heterogeneity (2 to 730 colonies/10(5) mononuclear cells), with some values indicating excessive self-renewal capacity of CFU-L. In contrast, 62% of the MDS specimens failed to produce any secondary colony growth, and PE2 in the remaining cases was low (5 to 99/10(5) MNC). We conclude that a different balance between self-renewal and determination could be responsible for a slower pace of clonal expansion in MDS, even if the proliferative activity of clonogenic cells is similar to that in AML.     

Effect of recombinant interferons on colony formation of blast progenitors in acute myeloblastic leukemia

The effect of recombinant interferons (rIFNs) on primary and secondary colony formation by blast progenitors (leukemic colony-forming units, L-CFU) from the peripheral blood of patients with acute myeloblastic leukemia (AML) was examined. rIFN-alpha, rIFN-beta, and rIFN-gamma inhibited L-CFU in a dose-dependent manner. When 3000 U/ml rIFN-alpha, -beta, and -gamma were added, L-CFU were suppressed to 20%, 12%, and 40% of the control cultures, respectively. The concentrations of rIFN-alpha, -beta, and -gamma required for 50% inhibition of colony formation were 63, 29, and 250 U/ml, respectively. The self-renewal capacity of L-CFU was inhibited by rIFNs in a dose-dependent manner, the degree of inhibition being the same for all three rIFNs. These rIFNs also inhibited normal granulocyte-macrophage progenitors to a similar degree as L-CFU. Taken together, these in vitro studies indicate that these rIFNs may be efficacious in the treatment of AML, though development of granulocytopenia may be observed as a complication of IFN therapy.     

The proliferation in suspension of the progenitors of the blast cells in acute myeloblastic leukemia

A minority of blast cells in acute myeloblastic leukemia (AML) form colonies in culture in methylcellulose when stimulated by media conditioned by normal leukocytes in the presence of phytohemagglutinin (PHA-LCM). Blast colonies can be replated successfully, either as pooled cells or suspensions from single colonies. However, the plating efficiency declines with repeated passages, and more than four subcultures have not been achieved. In this study, blast populations were cultured in suspension, with fetal calf serum, alpha-minimal essential medium and PHA-LCM. In cells from 17 of 18 patients, exponential growth of clonogenic blast cells was maintained for six to seven days without reculturing. Colonies obtained from progenitors taken from liquid culture and replated in methylcellulose were replated to obtain the secondary plating efficiency (PE2). In 14 cases, this value was maintained or increased. In three instances, PE2 fell following culture in methylcellulose. When cells in suspension were recultured, exponential growth continued. In nine instances, exponential growth was maintained for from seven to 70 days. During this time, PE2 was maintained. Results from experiments using velocity sedimentation separation and analysis of single colonies were consistent with the view that the increase in clonogenic cells in suspension was a manifestation of their self-renewal capacity. The observations also support a model of blast progenitor growth that contains the postulate that these are capable not only of self-renewal but also of determination-like events leading to loss of proliferative capacity.     

Inhibition of the self-renewal capacity of blast progenitors from acute myeloblastic leukemia patients by site-selective 8-chloroadenosine 3'',5''-cyclic monophosphate.

The physiologic balance between the two regulatory subunit isoforms, RI and RII, of cAMP-dependent protein kinase is disrupted in cancer cells; growth arrest and differentiation of malignant cells can be achieved when the normal ratio of these intracellular signal transducers of cAMP is restored by the use of site-selective cAMP analogs. In this study we evaluated the effects of the site-selective cAMP analog 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP) on clonogenic growth of blast progenitors from 15 patients with acute myeloblastic leukemia and 3 patients affected by advanced myelodysplastic syndrome. Leukemic blast progenitors undergo terminal divisions, giving rise to colonies in methylcellulose. The self-renewal capacity of blast progenitors is conversely reflected in a secondary methylcellulose assay after exponential growth of clonogenic cells in suspension cultures. In all the samples tested, 8-Cl-cAMP, at micromolar concentrations (0.1-50 microM), suppressed in a dose-dependent manner both primary colony formation in methylcellulose and the recovery of clonogenic cells from suspension culture. Strikingly, in the samples from the entire group of patients, 8-Cl-cAMP was more effective in inhibiting the self-renewing clonogenic cells than the terminally dividing blast cells (P = 0.005). In addition, in four out of six cases studied, 8-Cl-cAMP was able to induce a morphologic and/or immunophenotypic maturation of leukemic blasts. An evident reduction of RI levels in fresh leukemic cells after exposure to 8-Cl-cAMP was also detected. Our results showing that 8-Cl-cAMP is a powerful inhibitor of clonogenic growth of leukemic blast progenitors by primarily suppressing their self-renewal capacity indicate that this site-selective cAMP analog represents a promising biological agent for acute myeloblastic leukemia therapy in humans.     

Comparison of the proliferative activity and sensitivity to cytosine arabinoside of leukemic blast progenitors in acute myeloblastic leukemia at diagnosis and in relapse.

To determine whether the biological characteristics of leukemic cells change after repeated chemotherapy, we compared the proliferative activity and drug sensitivity of leukemic blast progenitors in 7 patients with acute myeloblastic leukemia at diagnosis and in relapse. The proliferative activity of leukemic blast progenitors was assessed based on primary (PE1) and secondary (PE2) colony formation in methylcellulose culture and on the recovery of clonogenic cells in suspension culture. The effect of cytosine arabinoside (Ara-C) on leukemic blast progenitors was studied both in methylcellulose and in suspension cultures. PE1 and PE2 values varied among the patients. PE2 of 4 patients out of 7 patients became significantly higher in relapse than at diagnosis. The sensitivity to Ara-C of leukemic blast progenitors deteriorated in 5 patients in relapse. The results suggest that the biological nature in terms of proliferative activity and Ara-C sensitivity of leukemic blast progenitors may change in the clinical course after chemotherapy.     

Relationship between the in vitro sensitivity to cytosine arabinoside of blast progenitors and the outcome of treatment in acute myeloblastic leukaemia patients

The sensitivity to cytosine arabinoside (Ara-C) of blast progenitors from 22 acute myeloblastic leukaemia (AML) patients was studied in methylcellulose and suspension cultures. Primary colony-formation (PE1) in methylcellulose reflects the terminal divisions of blast progenitors, while secondary colony formation (PE2) in methylcellulose and the clonogenic cells recovery in suspension are considered to be based on the self-renewal of blast progenitors. In any patient, PE2 or clonogenic cells in suspension were more sensitive to Ara-C than PE1. The results indicate that Ara-C effectively suppresses not only terminal divisions but also self-renewal of blast progenitors D10 Ara-C value, the dose required to reduce survival to 10% of control, for PE1, PE2 and clonogenic cells in suspension showed marked patient-to-patient variation. No significant correlation was found between D10 Ara-C in methylcellulose or suspension culture and the response to treatment with a combination chemotherapy of 6-mercaptopurine, Ara-C and daunorubicin. However, a relapsed patient whose D10 values in methylcellulose and suspension cultures were very high showed poor response to a high-dose Ara-C protocol, where Ara-C was given alone at a high dose. The application of chemosensitivity test as a prediction of the clinical outcome may be dependent on the treatment protocol.     

Early effect of interferon on mouse leukemia cells cultivated in a chemostat.

Mouse interferon preparations inhibited the multiplication of mouse leukemia L 1210 cells cultivated under steady-state conditions in a chemostat. The use of this sensitive and controlled system led to the detection of a rapid inhibition in the incorporation of (3-H)thymidine into cellular acid-precipitable material 2 hr after the addition of interferon, whereas an effect on cell multiplication was not detected until 22 hr later. Interferon exerted only a transitory effect on the incorporation of (3-H)uridine into acid-precipitable material and no effect on the incorporation of 14-C-amino acids into cellular protein. It is suggested that the chemostat offers many advantages for the investigation of those physiologic factors or chemotherapeutic substances that modify cell division.     

Synergism between interleukin-6 and interleukin-3 in supporting proliferation of human hematopoietic stem cells: comparison with interleukin-1 alpha

Currently available evidence suggests that in the steady state, the majority of hematopoietic stem cells are dormant in cell cycle and reside in the so-called G0 period. Studies in our laboratory indicated that once a stem cell leaves G0, its subsequent proliferation requires the presence of interleukin-3 (IL-3). Recently it was reported that interleukin-1 (IL-1) may stimulate stem cells to become sensitive to IL- 3. In a separate study, we observed that interleukin-6 (IL-6, also known as B cell stimulatory factor-2/interferon beta 2) possesses synergism with IL-3, shortening the G0 period of murine hematopoietic stem cells. We report here that human IL-6 and IL-3 act synergistically in support of the proliferation of progenitors for human blast cell colonies and that IL-1 alpha reveals no synergism with IL-3 when tested against purified human marrow progenitors. Panned My-10+ human marrow cells were plated in culture and on day 14 of incubation, either IL-3, IL-6, IL-1 alpha or a combination of these factors was added to the cultures. Blast cell colony formation was analyzed daily between days 18 and 32 of culture. IL-6 or IL-1 alpha alone failed to support blast cell colony formation. In the presence of IL-3 alone, blast cell colonies continued to emerge between days 21 and 27. When a combination of IL-3 and IL-6 was added, blast cell colonies developed earlier than in cultures with IL-3 alone and twice as many blast cell colonies were identified. IL-1 alpha failed to augment IL-3-dependent blast cell colony formation. Replating studies of the individual blast cell colonies revealed various types of single as well as multilineage colonies. These observations suggest that IL-6 shortens the G0 period of human hematopoietic stem cells and that the reported synergistic activities of IL-1 on primitive hematopoietic cells may be indirect.     

Ethanol-induced Inhibition of Liver Cell Function: I. Effect of Ethanol on Hormone Stimulated Hepatocyte DNA Synthesis and the Role of Ethanol Metabolism

We have studied a short term effect (96 hr) of ethanol on hormone-stimulated DNA synthesis in a primary rat hepatocyte culture system. Studies were also performed with respect to total RNA and protein synthesis as well as albumin secretion measured by a solid-phase radioimmunoassay. We found that ethanol, when added to cultured hepatocytes, resulted in a substantial reduction in hormone-stimulated hepatocyte DNA synthesis and this effect was concentration dependent and occurred in serum-free medium. Ethanol also had an inhibitory effect on total RNA synthesis but protein synthesis and albumin secretion remained essentially unchanged. We determined that hepatocytes exposed to ethanol during the first 24 hr of culture were the most susceptible to inhibition of DNA synthesis. During the first 24 hr, alcohol dehydrogenase (ADH) activity was present in the cells at higher levels than at 48 and 72 hr. In human hepatoma cell lines and differentiated primary and secondary chick fibroblasts, no ADH activity was demonstrable; such cells were not inhibited by 100 mM ethanol additions and DNA synthesis rates were similar to untreated cultures. Other alcohols found to be metabolized by hepatocyte ADH were inhibitory towards hormone-stimulated DNA synthesis whereas those with less metabolism had little effect. Hepatocytes treated with 4-methylpyrazole, an inhibitor of ADH, were partially protected from ethanol effects. Taken together our results are consistent with the hypothesis that a major physiological effect of ethanol on the hepatocyte is a direct impairment of DNA synthesis and that alcohol metabolism is required.     

Effects of granulocyte-macrophage colony-stimulating factor and erythropoietin on leukemic erythroid colony formation in human early erythroblastic leukemias

Erythroid colonies from five patients with an early erythroblastic leukemia were obtained in "serum-free" cultures in the presence or absence of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and homogeneous native erythropoietin (Epo). Erythroid colonies with abnormal morphology and karyotype could be grown in different culture conditions. Their erythroid nature was ascertained by the presence of carbonic anhydrase I and glycophorin A. Leukemic erythroid progenitors strongly differed from normal progenitors in that spontaneous colonies were always obtained, sometimes with an extremely high plating efficiency (up to 5.7%). Colonies were found to be autonomous from exogenous hematopoietic growth factors because they were still obtained with a high plating efficiency at an average of one cell per culture in the absence of any added growth factor. No evidence for an autocrine secretion of Epo or GM-CSF emerged because Epo or GM- CSF could not be detected by biologic or radioimmunologic assays from the culture supernatant or cellular extracts of the leukemic cells and that Epo or GM-CSF antibodies did not block autonomous growth. In all cases, however, hematopoietic growth factors increased the plating efficiency of the abnormal erythroid progenitors. In the two "de novo" leukemias, leukemic erythroid progenitors responded primarily to Epo, whereas in the three other patients' (chronic myeloid leukemia) blast crisis they responded maximally to GM-CSF plus Epo. Recombinant erythroid-potentiating activity had no effect in any of these cases. These results suggest that the leukemic erythroid clonogenic cells arise from expansion of erythroid progenitors at different levels of differentiation (ie, CFU-E or BFU-E, depending upon the disease) and that autonomous growth is not related to a secretion of Epo or GM-CSF.     

Interleukin 6 enhancement of interleukin 3-dependent proliferation of multipotential hemopoietic progenitors.

Interleukin-6 (IL-6, also known as B-cell stimulatory factor 2/interferon beta 2) was previously shown to support the proliferation of granulocyte/macrophage progenitors and indirectly support the formation of multilineage and blast cell colonies in cultures of spleen cells from normal mice. We report here that IL-3 and IL-6 act synergistically in support of the proliferation of murine multipotential progenitors in culture. The time course of total colony formation by spleen cells isolated from mice 4 days after injection of 5-fluorouracil (150 mg/kg) was significantly shortened in cultures containing both lymphokines relative to cultures supported by either of the two factors. Serial observations (mapping) of individual blast cell colonies in culture revealed that blast cell colonies emerged after random time intervals in the presence of IL-3. The average time of appearance in IL-6 alone was somewhat delayed, and in cultures containing both factors the appearance of multilineage blast cell colonies was significantly hastened relative to cultures grown in the presence of the individual lymphokines. In cultures of day-2 post-5-fluorouracil bone marrow cells, IL-6 failed to support colony formation; IL-3 alone supported the formation of a few granulocyte/macrophage colonies, but the combination of factors acted synergistically to yield multilineage and a variety of other types of colonies. In this system, IL-1 alpha also acted synergistically with IL-3, but the effect was smaller, and no multilineage colonies were seen. Together these results indicate that IL-3 and IL-6 act synergistically to support the proliferation of hemopoietic progenitors and that at least part of the effect results from a decrease in the G0 period of the individual stem cells.     

Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor.

Multiple rounds of cell division were induced in primary cultures of rat hepatocytes in serum-free medium containing 10 mmol/L nicotinamide and 10 ng epidermal growth factor/ml. Cells per culture almost doubled between day 1 and day 5. The proliferating cells were predominantly mononucleate. The time course of DNA synthesis in cultured hepatocytes showed that peaks of the incorporation of 3H-thymidine were observed at 60 hr and 82 hr after plating. Labeling indices of the cells indicated that almost half the cells were labeled with 3H-thymidine in the periods 48 to 72 hr and 72 to 96 hr after plating. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 hr in culture, as demonstrated by the use of continuous treatments with 3H-thymidine and 5-bromo-2'-deoxyuridine. Furthermore, by day 4 of culture, about 40% and 15% of metaphases resulted from a second and third round of cell division, respectively. The cultured hepatocytes on day 5 stained with albumin immunocytochemically, and the activity of tyrosine aminotransferase was induced by dexamethasone and glucagon on day 3. In addition, electron micrographs revealed that dividing cells not only had many characteristics of liver mitochondria and bile canaliculus-like structures, but many also contained a few large peroxisomes with internal crystalline nucleoids.     

Inhibition of acute myelogenous leukemia blast proliferation by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors.

Interleukin-1 (IL-1) has recently been reported to play an important role in acute myelogenous leukemia (AML) blast proliferation. We therefore investigated the effect of soluble IL-1 receptors (sIL-1R) and IL-1 receptor antagonist (IL-1RA) on the growth of AML bone marrow blast progenitors from 25 patients. In the AML blast colony culture assay, sIL-1R and IL-1RA inhibited blast colony-forming cell replication in a dose-dependent fashion, at concentrations ranging from 10 to 500 ng/mL (sIL-1R) and 10 to 1,000 ng/mL (IL-1RA), and their inhibitory effect was partially reversed by IL-1 beta. A similar inhibitory effect was also noted with the use of anti-IL-1 beta neutralizing antibodies. When AML blast progenitors were grown either in the presence of fetal calf serum (FCS) alone or with one of the following: phytohemagglutinin leukocyte-conditioned medium (PHA-LCM), granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, interleukin-3 (IL-3), or stem cell factor (SCF), addition of 100 ng/mL sIL-1R or IL-1RA inhibited blast colony formation by 3% to 96% and 2% to 97%, respectively. In sharp contrast, neither of these IL-1-inhibitory molecules significantly inhibited proliferation of normal marrow hematopoietic progenitors. Lysates of 2 x 10(7) low-density AML marrow cells were tested for intrinsic IL-1 beta content using an enzyme-linked immunoadsorbant assay (ELISA). Samples from five of six patients showed high concentrations (ranging from 501 to 2,041 pg), whereas 2 x 10(7) cells from two normal marrow aspirates yielded 54.6 pg of IL-1 beta. AML blast colony-forming cells from all six patients were inhibited by sIL-1R, IL-1RA, or both. Incubation of nine samples of AML low-density cells with either sIL-1R or IL-1RA reduced GM-CSF concentrations in cell lysates, and supernatants from nine (P less than .01) and six samples (P less than .037), respectively, and G-CSF concentration in lysates from six of nine samples (P less than .03), and in supernatants from five of six samples (P less than .06) when studied by ELISAs. Our data implicate IL-1 in AML blast proliferation and suggest the potential benefits of using IL-1-inhibitory molecules in future therapies for AML.     

Correlation between nicotine-induced inhibition of hematopoiesis and decreased CD44 expression on bone marrow stromal cells.

This study demonstrates that in vivo exposure to cigarette smoke (CS) and in vitro treatment of long-term bone marrow cultures (LTBMCs) with nicotine, a major constituent of CS, result in inhibition of hematopoiesis. Nicotine treatment significantly delayed the onset of hematopoietic foci and reduced their size. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) within an adherent layer of LTBMCs was significantly reduced in cultures treated with nicotine. Although the production of nonadherent mature cells and their progenitors in nicotine-treated LTBMCs was inhibited, this treatment failed to influence the proliferation of committed hematopoietic progenitors when added into methylcellulose cultures. Bone marrow stromal cells are an integral component of the hematopoietic microenvironment and play a critical role in the regulation of hematopoietic stem cell proliferation and self-renewal. Exposure to nicotine decreased CD44 surface expression on primary bone marrow-derived fibroblastlike stromal cells and MS-5 stromal cell line, but not on hematopoietic cells. In addition, mainstream CS altered the trafficking of hematopoietic stem/progenitor cells (HSPC) in vivo. Exposure of mice to CS resulted in the inhibition of HSPC homing into bone marrow. Nicotine and cotinine treatment resulted in reduction of CD44 surface expression on lung microvascular endothelial cell line (LEISVO) and bone marrow-derived (STR-12) endothelial cell line. Nicotine treatment increased E-selectin expression on LEISVO cells, but not on STR-12 cells. These findings demonstrate that nicotine can modulate hematopoiesis by affecting the functions of the hematopoiesis-supportive stromal microenvironment, resulting in the inhibition of bone marrow seeding by LTC-ICs and interfering with stem cell homing by targeting microvascular endothelial cells.