Exploration of Hydrazide-Based HDAC8 PROTACs for the Treatment of Hematological Malignancies and Solid Tumors.

HDAC8 can mediate signals by using its enzymatic or nonenzymatic functions, which are expected to be critical for various types of cancer. Herein, we employed proteolysis targeting chimera (PROTAC) technology to target the enzymatic as well as the nonenzymatic functions of HDAC8. A potent and selective HDAC8 PROTAC Z16 (CZH-726) with low nanomolar DC50 values in various cell lines was identified. Interestingly, Z16 induced structural maintenance of chromosomes protein 3 (SMC3) hyperacetylation at low concentrations and histone hyperacetylation at high concentrations, which can be explained by HDAC8 degradation and off-target HDAC inhibition, respectively. Notably, Z16 potently inhibited proliferation of various cancer cell lines and the antiproliferative mechanisms proved to be cell-type-dependent, which, to a large extent, is due to off-target HDAC inhibition. In conclusion, we report a hydrazide-based HDAC8 PROTAC Z16, which can be used as a probe to investigate the biological functions of HDAC8.

Discovery of highly potent HDAC8 PROTACs with anti-tumor activity.

Various diseases are deeply associated with aberrations in HDAC8 functions. These aberrations can be assigned to either structural functions or catalytic functions of HDAC8. Therefore, development of HDAC8 degradation inducers might be more promising than HDAC8 inhibitors. We employed the proteolysis targeting chimera (PROTAC) strategy to develop a selective and potent HDAC8 degradation inducer CT-4 with single-digit nanomolar DC50 values and over 95% Dmax in both triple-negative breast cancer MDA-MB-231 cells and T-cell leukemia cells. Notably, CT-4 demonstrated potent anti-migration activity and limited anti-proliferative activity in MDA-MB-231 cells. In contrast, CT-4 effectively induced apototic cell death in Jurkat cells, as assessed by a caspase 3/7 activity assay and flow cytometry. Our findings suggest that the development of HDAC8 degradation inducers holds great potential for the treatment of HDAC8-related diseases.

Receptor Specificity Engineering of TNF Superfamily Ligands.

The tumor necrosis factor (TNF) ligand family has nine ligands that show promiscuity in binding multiple receptors. As different receptors transduce into diverse pathways, the study on the functional role of natural ligands is very complex. In this review, we discuss the TNF ligands engineering for receptor specificity and summarize the performance of the ligand variants in vivo and in vitro. Those variants have an increased binding affinity to specific receptors to enhance the cell signal conduction and have reduced side effects due to a lowered binding to untargeted receptors. Refining receptor specificity is a promising research strategy for improving the application of multi-receptor ligands. Further, the settled variants also provide experimental guidance for engineering receptor specificity on other proteins with multiple receptors.

Identification of a Bromodomain-like Region in 15-Lipoxygenase-1 Explains Its Nuclear Localization.

Lipoxygenase (LOX) activity provides oxidative lipid metabolites, which are involved in inflammatory disorders and tumorigenesis. Activity-based probes to detect the activity of LOX enzymes in their cellular context provide opportunities to explore LOX biology and LOX inhibition. Here, we developed Labelox B as a potent covalent LOX inhibitor for one-step activity-based labeling of proteins with LOX activity. Labelox B was used to establish an ELISA-based assay for affinity capture and antibody-based detection of specific LOX isoenzymes. Moreover, Labelox B enabled efficient activity-based labeling of endogenous LOXs in living cells. LOX proved to localize in the nucleus, which was rationalized by identification of a functional bromodomain-like consensus motif in 15-LOX-1. This indicates that 15-LOX-1 is not only involved in oxidative lipid metabolism, but also in chromatin binding, which suggests a potential role in chromatin modifications.

Dihydroartemisinin-Transferrin Adducts Enhance TRAIL-Induced Apoptosis in Triple-Negative Breast Cancer in a P53-Independent and ROS-Dependent Manner.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype independent of estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2. It has a poor prognosis and high recurrence. Due to its limited treatment options in the clinic, novel therapies are urgently needed. Single treatment with the death receptor ligand TRAIL was shown to be poorly effective. Recently, we have shown that artemisinin derivatives enhance TRAIL-induced apoptosis in colon cancer cells. Here, we utilized transferrin (TF) to enhance the effectiveness of dihydroartemisinin (DHA) in inducing cell death in TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and BT549). We found that the combination of DHA-TF and the death receptor 5-specific TRAIL variant DHER leads to an increase in DR5 expression in all four TNBC cell lines, while higher cytotoxicity was observed in MDA-MB-231, and MDA-MB-436. All the data point to the finding that DHA-TF stimulates cell death in TNBC cells, while the combination of DHA-TF with TRAIL variants will trigger more cell death in TRAIL-sensitive cells. Overall, DHA-TF in combination with TRAIL variants represents a potential novel combination therapy for triple-negative breast cancer.

Artemisinin Derivatives Stimulate DR5-Specific TRAIL-Induced Apoptosis by Regulating Wildtype P53.

Artemisinin derivatives, widely known as commercial anti-malaria drugs, may also have huge potential in treating cancer cells. It has been reported that artemisinin derivatives can overcome resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in liver and cervical cancer cells. In our study, we demonstrated that artesunate (ATS) and dihydroartemisinin (DHA) are more efficient in killing colon cancer cells compared to artemisinin (ART). ATS/DHA induces the expression of DR5 in a P53 dependent manner in HCT116 and DLD-1 cells. Both ATS and DHA overcome the resistance to DHER-induced apoptosis in HCT116, mainly through upregulating death receptor 5 (DR5). We also demonstrate that DHA sensitizes HCT116 cells to DHER-induced apoptosis via P53 regulated DR5 expression in P53 knockdown assays. Nevertheless, a lower effect was observed in DLD-1 cells, which has a single Ser241Phe mutation in the P53 DNA binding domain. Thus, the status of P53 could be one of the determinants of TRAIL resistance in some cancer cells. Finally, the combination treatment of DHA and the TRAIL variant DHER increases cell death in 3D colon cancer spheroid models, which shows its potential as a novel therapy.

Death Receptor 5 Displayed on Extracellular Vesicles Decreases TRAIL Sensitivity of Colon Cancer Cells.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is considered to be a promising antitumor drug because of its selective proapoptotic properties on tumor cells. However, the clinical application of TRAIL is until now limited because of the resistance of several cancer cells, which can occur at various levels in the TRAIL signaling pathway. The role of decoy receptors that can side-track TRAIL, thereby preventing the formation of an activated death receptor, has been extensively studied. In this study, we have focused on extracellular vesicles (EVs) that are known to play a role in cell-to-cell communication and that can be released by donor cells into the medium transferring their components to recipient cells. TRAIL-induced apoptotic signaling is triggered upon the binding of two death receptors, DR4 and DR5. Here, we found that DR5 but not DR4 is present in the conditioned medium (CM)-derived from various cancer cells. Moreover, we observed that DR5 was exposed on EVs and can act as "decoy receptor" for binding to TRAIL. This results in a strongly reduced number of apoptotic cells upon treatment with DR5-specific TRAIL variant DHER in CM. This reduction happened with EVs containing either the long or short isoform of DR5. Taken together, we demonstrated that colon rectal tumor cells can secrete DR5-coated EVs, and this can cause TRAIL resistance. This is to our knowledge a novel finding and provides new insights into understanding TRAIL sensitivity.

Histone Deacetylase Inhibitors Sensitize TRAIL-Induced Apoptosis in Colon Cancer Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as a promising anti-cancer therapeutic. However, many cancers have been found to be or to become inherently resistant to TRAIL. A combination of epigenetic modifiers, such as histone deacetylase inhibitors (HDACi's), with TRAIL was effective to overcome TRAIL resistance in some cancers. Broad spectrum HDACi's, however, show considerable toxicity constraining clinical use. Since overexpression of class I histone deacetylase (HDAC) has been found in colon tumors relative to normal mucosa, we have focused on small spectrum HDACi's. We have now tested agonistic receptor-specific TRAIL variants rhTRAIL 4C7 and DHER in combination with several class I specific HDACi's on TRAIL-resistant colon cancer cells DLD-1 and WiDr. Our data show that TRAIL-mediated apoptosis is largely improved in WiDr cells by pre-incubation with Entinostat-a HDAC1, 2, and 3 inhibitor- and in DLD-1 cells by RGFP966-a HDAC3-specific inhibitor- or PCI34051-a HDAC8-specific inhibitor. We are the first to report that using RGFP966 or PCI34051 in combination with rhTRAIL 4C7 or DHER represents an effective cancer therapy. The intricate relation of HDACs and TRAIL-induced apoptosis was confirmed in cells by knockdown of HDAC1, 2, or 3 gene expression, which showed more early apoptotic cells upon adding rhTRAIL 4C7 or DHER. We observed that RGFP966 and PCI34051 increased DR4 expression after incubation on DLD-1 cells, while RGFP966 induced more DR5 expression on WiDr cells, indicating a different role for DR4 or DR5 in these combinations. At last, we show that combined treatment of RGFP966 with TRAIL variants (rhTRAIL 4C7/DHER) increases apoptosis on 3D tumor spheroid models.

Creation of RANKL mutants with low affinity for decoy receptor OPG and their potential anti-fibrosis activity.

Fibrosis is characterized by the progressive alteration of the tissue structure due to the excessive production of extracellular matrix (ECM). The signaling system encompassing Receptor Activator of Nuclear factor NF-κB Ligand (RANKL)/RANK/Osteoprotegerin (OPG) was discovered to play an important role in the regulation of ECM formation and degradation in bone tissue. However, whether and how this signaling pathway plays a role in liver or pulmonary ECM degradation is unclear up to now. Interestingly, increased decoy receptor OPG levels are found in fibrotic tissues. We hypothesize that RANKL can stimulate RANK on macrophages and initiate the process of ECM degradation. This process may be inhibited by highly expressed OPG in fibrotic conditions. In this case, RANKL mutants that can bind to RANK without binding to OPG might become promising therapeutic candidates. In this study, we built a structure-based library containing 44 RANKL mutants and found that the Q236 residue of RANKL is important for OPG binding. We show that RANKL_Q236D can activate RAW cells to initiate the process of ECM degradation and is able to escape from the obstruction by exogenous OPG. We propose that the generation of RANKL mutants with reduced affinity for OPG is a promising strategy for the exploration of new therapeutics against fibrosis.

CX Chemokine Receptor 7 Contributes to Survival of KRAS-Mutant Non-Small Cell Lung Cancer upon Loss of Epidermal Growth Factor Receptor.

KRAS-driven non-small cell lung cancer (NSCLC) patients have no effective targeted treatment. In this study, we aimed to investigate targeting epidermal growth factor receptor (EGFR) as a therapeutic approach in KRAS-driven lung cancer cells. We show that ablation of EGFR significantly suppressed tumor growth in KRAS-dependent cells and induced significantly higher expression of CX chemokine receptor 7 (CXCR7) and activation of MAPK (ERK1/2). Conversely, rescue of EGFR led to CXCR7 downregulation in EGFR-/- cells. Dual EGFR and CXCR7 inhibition led to substantial reduction of MAPK (pERK) and synergistic inhibition of cell growth. Analysis of two additional EGFR knockout NSCLC cell lines using CRISPR/Cas9 revealed genotype dependency of CXCR7 expression. In addition, treatment of different cells with gefitinib increased CXCR7 expression in EGFRwt but decreased it in EGFRmut cells. CXCR7 protein expression was detected in all NSCLC patient samples, with higher levels in adenocarcinoma as compared to squamous cell lung carcinoma and healthy control cases. In conclusion, EGFR and CXCR7 have a crucial interaction in NSCLC, and dual inhibition may be a potential therapeutic option for NSCLC patients.

Death receptor 5 is activated by fucosylation in colon cancer cells.

The remarkable pro-apoptotic properties of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) have led to considerable interest in this protein as a potential anticancer therapeutic. However, TRAIL is largely ineffective in inducing apoptosis in certain cancer cells, and the mechanisms underlying this selectivity are unknown. In colon adenocarcinomas, posttranslational modifications including O- and N- glycosylation of death receptors were found to correlate with TRAIL-induced apoptosis. Additionally, mRNA levels of fucosyltransferase 3 (FUT3) and 6 (FUT6) were found to be high in the TRAIL-sensitive colon adenocarcinoma cell line COLO 205. In this study, we use agonistic receptor-specific TRAIL variants to dissect the contribution of FUT3 and FUT6-mediated fucosylation to TRAIL-induced apoptosis via its two death receptors, DR4 and DR5. Triggering of apoptosis by TRAIL revealed that the low FUT3/6-expressing cells DLD-1 and HCT 116 are insensitive to DR5 but not to DR4-mediated apoptosis. By contrast, efficient apoptosis is mediated via both receptors in high FUT3/6-expressing COLO 205 cells. The reconstitution of FUT3/6 expression in DR5-resistant cells completely restored TRAIL sensitivity via this receptor, while only marginally enhancing apoptosis via DR4 at lower TRAIL concentrations. Interestingly, we observed that induction of the salvage pathway by external administration of l-fucose restores DR5-mediated apoptosis in both DLD-1 and HCT 116 cells. Finally, we show that fucosylation influences the ligand-independent receptor association that leads to increased death inducing signalling complex (DISC) formation and caspase-8 activation. Taken together, these results provide evidence for the differential impact of fucosylation on signalling via DR4 or DR5. These findings provide novel opportunities to enhance TRAIL sensitivity in colon adenocarcinoma cells that are highly resistant to DR5-mediated apoptosis.

PvdQ Quorum Quenching Acylase Attenuates Pseudomonas aeruginosa Virulence in a Mouse Model of Pulmonary Infection.

Pseudomonas aeruginosa is the predominant pathogen in pulmonary infections associated with cystic fibrosis. Quorum sensing (QS) systems regulate the production of virulence factors and play an important role in the establishment of successful P. aeruginosa infections. Inhibition of the QS system (termed quorum quenching) renders the bacteria avirulent thus serving as an alternative approach in the development of novel antibiotics. Quorum quenching in Gram negative bacteria can be achieved by preventing the accumulation of N-acyl homoserine lactone (AHL) signaling molecule via enzymatic degradation. Previous work by us has shown that PvdQ acylase hydrolyzes AHL signaling molecules irreversibly, thereby inhibiting QS in P. aeruginosa in vitro and in a Caenorhabditis elegans model of P. aeruginosa infection. The aim of the present study is to assess the therapeutic efficacy of intranasally instilled PvdQ acylase in a mouse model of pulmonary P. aeruginosa infection. First, we evaluated the deposition pattern of intranasally administered fluorochrome-tagged PvdQ (PvdQ-VT) in mice at different stages of pulmonary infection by in vivo imaging studies. Following intranasal instillation, PvdQ-VT could be traced in all lung lobes with 42 ± 7.5% of the delivered dose being deposited at 0 h post-bacterial-infection, and 34 ± 5.2% at 72 h post bacterial-infection. We then treated mice with PvdQ during lethal P. aeruginosa pulmonary infection and that resulted in a 5-fold reduction of lung bacterial load and a prolonged survival of the infected animals with the median survival time of 57 hin comparison to 42 h for the PBS-treated group. In a sublethal P. aeruginosa pulmonary infection, PvdQ treatment resulted in less lung inflammation as well as decrease of CXCL2 and TNF-α levels at 24 h post-bacterial-infection by 15 and 20%, respectively. In conclusion, our study has shown therapeutic efficacy of PvdQ acylase as a quorum quenching agent during P. aeruginosa infection.

Cytotoxic Deoxypodophyllotoxin Can Be Extracted in High Purity from Anthriscus sylvestris Roots by Supercritical Carbon Dioxide.

Deoxypodophyllotoxin is present in the roots of Anthriscus sylvestris. This compound is cytotoxic on its own, but it can also be converted into podophyllotoxin, which is in high demand as a precursor for the important anticancer drugs etoposide and teniposide. In this study, deoxypodophyllotoxin is extracted from A. sylvestris roots by supercritical carbon dioxide extraction. The process is simple and scalable. The supercritical carbon dioxide method extracts 75 - 80% of the total deoxypodophyllotoxin content, which is comparable to a single extraction by traditional Soxhlet. However, less polar components are extracted. The activity of the supercritical carbon dioxide extract containing deoxypodophyllotoxin was assessed by demonstrating that the extract arrests A549 and HeLa cells in the G2/M phase of the cell cycle. We conclude that biologically active deoxypodophyllotoxin can be extracted from A. sylvestris by supercritical carbon dioxide extraction. The method is solvent free and more sustainable compared to traditional methods.

Novel RANKL DE-loop mutants antagonize RANK-mediated osteoclastogenesis.

Bone is a dynamic tissue that is maintained by continuous renewal. An imbalance in bone resorption and bone formation can lead to a range of disorders, such as osteoporosis. The receptor activator of NF-κB (RANK)-RANK-ligand (RANKL) pathway plays a major role in bone remodeling. Here, we investigated the effect of mutations at position I248 in the DE-loop of murine RANKL on the interaction of RANKL with RANK, and subsequent activation of osteoclastogenesis. Two single mutants, RANKL I248Y and I248K, were found to maintain binding and have the ability to reduce wild-type RANKL-induced osteoclastogenesis. The generation of RANK-antagonists is a promising strategy for the exploration of new therapeutics against osteoporosis.

The ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells. In A172 cells, sub-toxic concentrations of DMC greatly potentiated TRAIL-induced apoptosis. Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis. Our findings corroborate that DMC is a promising agent against GBM, and uncovers a potential synergistic cooperation with TRAIL in this highly malignant cancer.

Erratum to: the ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma.

[This corrects the article DOI: 10.1186/2193-1801-3-495.].

Synthetic constrained peptide selectively binds and antagonizes death receptor 5.

Apoptosis or programmed cell death is an inherent part of the development and homeostasis of multicellular organisms. Dysregulation of apoptosis is implicated in the pathogenesis of diseases such as cancer, neurodegenerative diseases and autoimmune disorders. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce apoptosis by binding death receptor (DR)4 (TRAIL-R1) and DR5 (TRAIL-R2), which makes TRAIL an interesting and promising therapeutic target. To identify peptides that specifically interact with DR5, a disulfide-constrained phage display peptide library was screened for binders towards this receptor. Phage-displayed peptides were identified that bind specifically to DR5 and not to DR4, nor any of the decoy receptors. We show that the synthesized peptide, YCKVILTHRCY, in both monomeric and dimeric forms, binds specifically to DR5 in such a way that TRAIL binding to DR5 is inhibited. Surface plasmon resonance studies showed higher affinity towards DR5 for the dimeric form then the monomeric form of the peptide, with apparent K(d) values of 40 nm versus 272 nm, respectively. Binding studied on cell lines by flow cytometry analyses showed concentration-dependent binding. Upon co-incubation with increasing concentrations of TRAIL, the peptide binding was reduced. Moreover, both the monomeric and dimeric forms of the peptide reduced TRAIL-induced cell death in Colo205 colon carcinoma cells. The peptide, YCKVILTHRCY, or its derivates, may be a useful investigative tool for dissecting signalling via DR5 relative to DR4 or could act as a lead peptide for the development of therapeutic agents in diseases with dysregulated TRAIL-signalling.

Differences in heat sensitivity between normal and acute myeloid leukemic stem cells: feasibility of hyperthermic purging of leukemic cells from autologous stem cell grafts.

OBJECTIVES: In autologous stem cell transplantation contamination of the graft with malignant cells is frequently noticed and necessitates the use of in vivo or in vitro purging modalities. The hematopoietic recovery after transplantation depends on the number of stem and progenitor cells in the transplant. Therefore, in the present study the effects of hyperthermic treatment on the human normal and acute myeloid leukemic (AML) stem cell compartment were investigated. METHODS: Normal bone marrow and AML blasts were heat treated up to 120 minutes at 43 degrees C. The surviving fractions of the different stem cell subsets were determined using in vitro methylcellulose and cobblestone area-forming cell (CAFC) clonogenic assays, as well as the in vivo NOD/SCID repopulating assay. The leukemic nature of the colonies from AML cells was confirmed by RT-PCR analysis. In order to increase the therapeutic index of the hyperthermic purging modality, the heat treatment was preceded by a 3-hour incubation at 37 degrees C with the ether lipid ET-18-OCH(3) (25 microg/mL). RESULTS: It could be demonstrated that normal progenitor cells are far more resistant to hyperthermia than leukemic progenitor cells (56%+/-7% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). Furthermore, normal hematopoietic stem cells appear to be extremely resistant to the heat treatment (94%+/-9% survival after 60 minutes at 43 degrees C). In contrast, in the leukemic stem cell compartment no significant differences in heat sensitivity between the stem cells and progenitor subsets could be observed (12.3%+/-2.9% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). The combined treatment resulted in a survival for normal progenitor and stem cells of 32%+/-6% and 85%+/-15% after 60 minutes at 43 degrees C, respectively. Under these conditions the number of leukemic stem cells was reduced to 1%+/-0.3%. After 120 minutes at 43 degrees C, no AML-colonies could be detected anymore. CONCLUSIONS: Our data demonstrate that leukemic stem cells have an increased hyperthermic sensitivity compared to their normal counterparts and that this difference can be further increased in combination with ET-18-OCH(3). These striking differences in heat sensitivity warrant the use of hyperthermia as a clinically applicable purging modality in autologous stem cell transplantation.

Peripheral blood stem cells differ from bone marrow stem cells in cell cycle status, repopulating potential, and sensitivity toward hyperthermic purging in mice mobilized with cyclophosphamide and granulocyte colony-stimulating factor.

Peripheral blood stem cells (PBSCs) are increasingly used in autologous stem cell transplantations. We investigated the mobilizing effect of a combined cyclophosphamide (CTX) and granulocyte colony-stimulating factor (G-CSF) treatment on progenitor cells (STRA) and primitive stem cells (LTRA) in normal and splenectomized CBA/H mice. This combined treatment not only resulted in mobilization but also in expansion of hematopoietic stem cell subsets. The latter phenomenon was somewhat suppressed in splenectomized animals, but in these mice an enhanced mobilization of STRA and LTRA cells into the peripheral blood was observed. Furthermore, we studied the engraftment potential of mobilized PBSCs. Mice transplanted with PBSCs engrafted significantly better compared to mice transplanted with bone marrow stem cells from control and mobilized mice. The repopulation curve was characterized by a less-deep nadir indicating that the differences occur during the initial phase after transplantation. Contamination of autologous PBSC transplants with malignant cells is noticed frequently and is the basis for urging the use of purging modalities. Here we used hyperthermia and found that the mobilized progenitor cells in peripheral blood are more resistant to hyperthermia than those in the bone marrow (i.e., a survival of 11 +/- 5% after 90 min at 43 degrees C for peripheral blood progenitors, compared to 0.5 +/- 0.4% in bone marrow of mobilized animals and 1.6 +/- 0.5% in normal animals, respectively). Hyperthermic purging does not eliminate the superior repopulating features of a PBSC graft, as is demonstrated by an increased median survival time of lethally irradiated mice transplanted with purged PBSCs. In conclusion, our data demonstrate that CTX + G-CSF-mobilized PBSCs have an enhanced engraftment potential concomitantly with a decreased cycling activity and hence a decreased hyperthermic sensitivity. These findings support the use of these mobilized PBSCs for autologous stem cell transplantation and strengthen the basis for using hyperthermia as a purging modality.