Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy.

Myotonic dystrophy (DM) is an autosomal dominant disorder resulting from the expansion of a CTG repeat in the 3' untranslated region of a putative protein kinase (DMPK). To elucidate the role of DMPK in DM pathogenesis we have developed Dmpk deficient (Dmpk-/-) mice. Dmpk-/-mice develop a late-onset, progressive skeletal myopathy that shares some pathological features with DM. Muscles from mature mice show variation in fibre size, increased fibre degeneration and fibrosis. Adult Dmpk-/-mice show ultrastructural changes in muscle and a 50% decrease in force generation compared to young mice. Our results indicate that DMPK may be necessary for the maintenance of skeletal muscle structure and function and suggest that a decrease in DMPK levels may contribute to DM pathology.

Label-free critical micelle concentration determination of bacterial quorum sensing molecules.

A practical label-free method for the rapid determination of small-molecule critical micelle concentration (CMC) using a fixed-angle light-scattering technique is described. Change in 90° light scattering at a fixed wavelength of incident radiation with increasing bacterial quorum molecule concentration and the observation of a break point is used to determine CMC. In our study, this technique is utilized to investigate the aqueous CMC of previously uncharacterized Pseudomonas aeruginosa quorum sensing signaling molecules (QSSM) belonging to the n-acylhomoserine lactone and 2-alkyl-4-quinolone classes. Several were found to form micelles within a physiologically relevant concentration range and potential roles of these micelles as QSSM transporters are discussed. The influence of temperature and the presence of biological membranes or serum proteins on QSSM CMC are also investigated and evidence is obtained to suggest the QSSMs studied are capable of both membrane and serum protein interaction. This demonstrates that the fixed-angle light-scattering technique outlined can be used simply and rapidly to determine small-molecule CMC under a variety of conditions.

Research and development of a silicone letrozole-releasing device to control reproduction in cattle.

Two experiments were conducted to test the effectiveness of a silicone matrix as an intravaginal drug delivery device for letrozole, an aromatase inhibitor used for synchronization protocols in cattle. A wax dip-coat formulation of the intravaginal device used in previous studies was effective in releasing letrozole but was cumbersome to manufacture and deploy, resulting in unwanted variation in drug delivery and circulating concentrations of letrozole. In Experiment 1, a 3 × 3 design was used to test the release kinetics of letrozole from silicone in vitro. Silicone was mixed with 3 different letrozole drug loads (5%, 10%, 15%) and 3 different mineral oil loads (5%, 10%, 15%), and letrozole release into 62.5% ethanol was compared with the wax dip-coat formulation (positive control) by UV spectrophotometry. Letrozole was released from silicone in a dose-dependent manner, with no effect of mineral oil. Release kinetics were then examined in vivo (Experiment 2) in nulliparous beef heifers assigned randomly to six groups (n = 6/group) and given either a large surface area (LSA) with 5% or 15% drug load, a standard surface area (SSA) intravaginal silicone device impregnated with 10% or 15% drug load, a wax dip-coat device (positive control), or a blank device (negative control). Devices were inserted on Day 3 (Day 0 = ovulation) until Day 11. Blood samples were collected at 0, 30 min, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h, and twice daily until Day 11 to determine letrozole plasma concentrations by LC-MS/MS and estradiol plasma concentrations by radioimmunoassay The ovaries were examined once daily from Day 3-13 by ultrasonography to determine follicular and luteal responses to treatment. Letrozole plasma concentrations were higher in heifers given a device with an LSA vs SSA (P < 0.05). Plasma concentrations of estradiol decreased the most in heifers given the 15% LSA device (P = 0.06). The interval between emergence of successive follicular waves was longest (P < 0.05) in the positive control and the 15% LSA groups. As well, the diameter profiles of the dominant follicle and the corpus luteum were largest (P < 0.01) in the positive control and 15% LSA groups. In conclusion, letrozole was released from a silicone matrix in vitro in a dose-dependent manner, and the 15% LSA devices achieved target effects on ovarian function. Results may be used to manufacture a silicone intravaginal device for delivering aromatase inhibitors in a novel synchronization protocol for cattle.

Cutaneous overexpression of NT-3 increases sensory and sympathetic neuron number and enhances touch dome and hair follicle innervation.

Target-derived influences of nerve growth factor on neuronal survival and differentiation are well documented, though effects of other neurotrophins are less clear. To examine the influence of NT-3 neurotrophin overexpression in a target tissue of sensory and sympathetic neurons, transgenic mice were isolated that overexpress NT-3 in the epidermis. Overexpression of NT-3 led to a 42% increase in the number of dorsal root ganglia sensory neurons, a 70% increase in the number of trigeminal sensory neurons, and a 32% increase in sympathetic neurons. Elevated NT-3 also caused enlargement of touch dome mechanoreceptor units, sensory end organs innervated by slowly adapting type 1 (SA1) neurons. The enlarged touch dome units of the transgenics had an increased number of associated Merkel cells, cells at which SA1s terminate. An additional alteration of skin innervation in NT-3 transgenics was an increased density of myelinated circular endings associated with the piloneural complex. The enhancement of innervation to the skin was accompanied by a doubling in the number of sensory neurons expressing trkC. In addition, measures of nerve fibers in cross-sectional profiles of cutaneous saphenous nerves of transgenics showed a 60% increase in myelinated fibers. These results indicate that in vivo overexpression of NT-3 by the epidermis enhances the number of sensory and sympathetic neurons and the development of selected sensory endings of the skin.

Convergence of the secretory pathways for cholera toxin and the filamentous phage, CTXphi.

Virulence of Vibrio cholerae depends on secretion of cholera toxin (CT), which is encoded within the genome of a filamentous phage, CTXphi. Release of CT is mediated by the extracellular protein secretion (eps) type II secretion system. Here, the outer membrane component of this system, EpsD, was shown to be required for secretion of the phage as well. Thus, EpsD plays a role both in pathogenicity and in horizontal transfer of a key virulence gene. Genomic analysis suggests that additional filamentous phages also exploit chromosome-encoded outer membrane channels.

Plasma homovanillic acid concentration and the severity of schizophrenic illness.

Concentrations of plasma homovanillic acid before treatment were highly correlated with global severity of illness in schizophrenic patients, both before and after treatment. In contrast, a fixed dose of haloperidol did not affect those concentrations. Thus, in patients with a diagnosis of schizophrenia, plasma homovanillic acid may reflect the severity of illness, but not be influenced by short-term pharmacological perturbations by neuroleptics.

Simultaneous co-delivery of neuroprotective drugs from multi-loaded PLGA microspheres for the treatment of glaucoma.

Glaucoma is a multifactorial neurodegenerative disorder and one of the leading causes of irreversible blindness globally and for which intraocular pressure is the only modifiable risk factor. Although neuroprotective therapies have been suggested to have therapeutic potential, drug delivery for the treatment of ocular disorders such as glaucoma remains an unmet clinical need, further complicated by poor patient compliance with topically applied treatments. In the present study we describe the development of multi-loaded PLGA-microspheres (MSs) incorporating three recognised neuroprotective agents (dexamethasone (DX), melatonin (MEL) and coenzyme Q10 (CoQ10)) in a single formulation (DMQ-MSs) to create a novel sustained-release intraocular drug delivery system (IODDS) for the treatment of glaucoma. MSs were spherical, with a mean particle size of 29.04 ±â€¯1.89 µm rendering them suitable for intravitreal injection using conventional 25G-32G needles. >62% incorporation efficiency was achieved for the three drug cargo and MSs were able to co-deliver the encapsulated active compounds in a sustained manner over 30-days with low burst release. In vitro studies showed DMQ-MSs to be neuroprotective in a glutamate-induced cytotoxicity model (IC50 10.00 ±â€¯0.94 mM versus 6.89 ±â€¯0.82 mM in absence of DMQ-MSs) in R28 cell line. In vivo efficacy studies were performed using a well-established rodent model of chronic ocular hypertension (OHT), comparing single intravitreal injections of microspheres of DMQ-MSs to their equivalent individual single-drug loaded MSs mixture (MSsmix), empty MSs, no-treatment OHT only and naïve groups. Twenty one days after OHT induction, DMQ-MSs showed a significantly neuroprotective effect on RGCs compared to OHT only controls. No such protective effect was observed in empty MSs and single-drug MSs treated groups. This work suggests that multi-loaded PLGA MSs present a novel therapeutic approach in the management of retinal neurodegeneration conditions such as glaucoma.

p21(Waf1/Cip1/Sdi1) mediates retinoblastoma protein degradation.

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21(Waf1) (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.

Transient receptor potential vanilloid 1-immunopositive neurons in the mouse are more prevalent within colon afferents compared to skin and muscle afferents.

Previous studies in our laboratories found that isolectin B(4)(IB(4))-positive polymodal nociceptors in the mouse do not express transient receptor potential vanilloid 1 (TRPV1), nor does deletion of TRPV1 compromise the ability of these afferents to detect thermal stimuli. Considering that IB(4)-positive afferents account for over 70% of cutaneous nociceptors and that 30-50% of all mouse primary afferents express TRPV1, it is highly likely that many TRPV1-positive fibers project to non-cutaneous structures. To investigate this issue, Alexa Fluor-conjugated wheat germ agglutinin (WGA) or IB(4) was injected into the nerves innervating quadriceps muscle (femoral) or hindlimb skin (saphenous) of male C57Bl/6 mice. Similarly, Alexa Fluor-conjugated cholera toxin-beta was injected subserosally into the distal colon. Spinal ganglia at the appropriate level (L2-3 for saphenous and femoral nerves; L6 for colon) were processed for TRPV1, calcitonin gene-related peptide (CGRP), neurofilament heavy chain (NHF) and IB(4) visualization and examined on a confocal microscope. Colon afferents contained the highest percentage of both TRPV1- and CGRP-positive neurons, followed by femoral (WGA) and saphenous afferents (WGA and IB(4)). In contrast, NHF staining was more prevalent among femoral afferents, followed by saphenous (WGA) and colon afferents. IB(4) binding was observed in very few colon or saphenous (WGA) afferents, with no femoral afferents binding or transporting IB(4). Considering that the largest percentages of TRPV1-positive neurons observed in this study were within visceral and muscle afferent populations (neurons that typically are not subject to noxious temperatures), these results suggest that TRPV1 may not function primarily as a temperature sensor but rather as a detector of protons, vanilloid compounds or through interactions with other membrane proteins.

Induction of growth factor RNA expression in human malignant melanoma: markers of transformation.

Alteration in the expression of growth factors is widely accepted as being one of several critical defects in the generation of the malignant cell. In the present study, 19 human metastatic melanoma cell lines were compared to 14 normal human foreskin melanocyte cell lines for the production of RNA transcripts specific for 11 different growth factors. Using the extremely sensitive technique of polymerase chain reaction to amplify growth factor-specific complementary DNAs, we analyzed the following: transforming growth factor (TGF) types alpha, beta 1, beta 2, and beta 3, acidic (a) fibroblast growth factor (FGF), basic (b) FGF, FGF-5, keratinocyte growth factor (KGF), HST, and platelet-derived growth factor (PDGF) types A and B. There were clear distinctions among the patterns of growth factor RNA expression by normal melanocytes and malignant melanoma cells. The prototypic melanocyte pattern of expression included TGF beta 1, TGF beta 3, and KGF. A subset of melanocyte cell lines also expressed PDGFA transcripts. In contrast, melanoma cells characteristically expressed RNA transcripts of TGF beta 1, TGF beta 2, TGF beta 3, TGF alpha, bFGF, KGF, and PDGFA. Subsets of melanoma cell lines also expressed aFGF, FGF-5, and PDGFB. The results presented indicated that TGF beta 2, TGF alpha, and bFGF may be particularly important in melanomagenesis and that these, as well as FGF-5, aFGF, and PDGFB, can be used as markers of transformation in this tumor type.

Selection for G156A O6-methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and protection from lethality in mice treated with O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea.

A retroviral gene therapy approach was developed to protect early hematopoietic progenitors from 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a stem cell toxin, and O6-benzylguanine (BG), an inhibitor of a key BCNU resistance protein, O6-alkylguanine DNA alkyltransferase (AGT). The retroviral vector MFG was used to transfer the G156A MGMT (deltaMGMT) cDNA, encoding a mutant AGT that is resistant to inhibition by BG, into murine bone marrow-derived hematopoietic progenitors. Following transplantation into lethally irradiated mice, the transduced cells were subjected to in vivo BG and BCNU treatment to examine the ability to enrich for transduced cells expressing deltaAGT. Transplantation of deltaMGMT-transduced cells resulted in deltaAGT expression in 30% of bone marrow nucleated cells 13 weeks after transplantation. After one cycle of BG and BCNU, deltaAGT expression was observed in 60% of bone marrow cells, and the percentage of colony-forming units (culture; CFU-C) containing proviral sequence increased from 67 to 100%. CFU-C obtained from BG and BCNU-treated deltaMGMT animals up to 23 weeks after transplantation were more resistant to combination BG and BCNU than CFU-C from mice transplanted with lacZ-transduced cells and treated with BG and BCNU or from mice transplanted with deltaMGMT-transduced cells and left untreated. The degree of drug resistance in deltaMGMT-transduced hematopoietic progenitors to BG and BCNU was much greater than we observed previously with wild-type MGMT gene transfer and treatment with BCNU alone. Furthermore, whereas 21 of 22 mice transplanted with deltaMGMT-transduced cells survived in vivo BG and BCNU administration, only 3 of 13 mice transplanted with lacZ-transduced progenitors survived similar drug treatment. Thus, deltaMGMT-transduced murine bone marrow cells selectively survive in vivo BG and BCNU exposure, resulting in prolonged enrichment for the transduced cells and protection from mortality induced by this drug combination.

Automatic quantitative analysis of experimental primary and secondary retinal neurodegeneration: implications for optic neuropathies.

Secondary neurodegeneration is thought to play an important role in the pathology of neurodegenerative disease, which potential therapies may target. However, the quantitative assessment of the degree of secondary neurodegeneration is difficult. The present study describes a novel algorithm from which estimates of primary and secondary degeneration are computed using well-established rodent models of partial optic nerve transection (pONT) and ocular hypertension (OHT). Brn3-labelled retinal ganglion cells (RGCs) were identified in whole-retinal mounts from which RGC density, nearest neighbour distances and regularity indices were determined. The spatial distribution and rate of RGC loss were assessed and the percentage of primary and secondary degeneration in each non-overlapping segment was calculated. Mean RGC number (82 592±681) and RGC density (1695±23.3 RGC/mm(2)) in naïve eyes were comparable with previous studies, with an average decline in RGC density of 71±17 and 23±5% over the time course of pONT and OHT models, respectively. Spatial analysis revealed greatest RGC loss in the superior and central retina in pONT, but significant RGC loss in the inferior retina from 3 days post model induction. In comparison, there was no significant difference between superior and inferior retina after OHT induction, and RGC loss occurred mainly along the superior/inferior axis (~30%) versus the nasal-temporal axis (~15%). Intriguingly, a significant loss of RGCs was also observed in contralateral eyes in experimental OHT. In conclusion, a novel algorithm to automatically segment Brn3a-labelled retinal whole-mounts into non-overlapping segments is described, which enables automated spatial and temporal segmentation of RGCs, revealing heterogeneity in the spatial distribution of primary and secondary degenerative processes. This method provides an attractive means to rapidly determine the efficacy of neuroprotective therapies with implications for any neurodegenerative disorder affecting the retina.

Overexpression of nerve growth factor in skin selectively affects the survival and functional properties of nociceptors.

Mice that overexpress nerve growth factor (NGF-OE) in the skin have double the normal number of cutaneous sensory neurons, have increased innervation of the skin and spinal cord, and are hyperalgesic. Here, we have asked whether the increased cutaneous NGF level results in a selective survival of only certain functional types of neurons and whether it changes the properties of cutaneous neurons. Using electron microscopy, we show that the number of both myelinated and unmyelinated nociceptors increases substantially in NGF-OE mice by a factor of 3.3 and 1.5, respectively. Using extracellular recordings from single units, we demonstrate that large myelinated (Abeta) fibers are unchanged in prevalence and receptive properties. In contrast, among thin myelinated (Adelta) fibers, the percentage of nociceptors increased from a normal 65 to 97%, consistent with a selective survival of nociceptors during embryogenesis. These afferents showed a twofold increase in their mechanical responsiveness, but their heat responsiveness remained normal. Among unmyelinated (C) fibers, there was a profound increase in the percentage of heat responsive neurons from a normal 42 to 96%. This change cannot be accounted for by a selective survival of heat-sensitive neurons. Unmyelinated nociceptors increased fourfold in their thermal responsiveness but decreased in mechanical responsiveness. Therefore, target-derived NGF selectively rescues nociceptors during the period of programmed cell death with different efficacy for thin myelinated or unmyelinated fibers. NGF also affects the response to noxious heat or mechanical stimuli in each group differently, implying specific regulations of transduction processes rather than general changes of excitability.

Overexpression of brain-derived neurotrophic factor enhances sensory innervation and selectively increases neuron number.

Target-derived neurotrophin growth factors have significant effects on the development and maintenance of the mammalian somatosensory system. Studies of transgenic mice that overexpress neurotrophins NGF and neurotrophin 3 (NT-3) at high levels in skin have shown increased sensory neuron number and enhanced innervation of specific sensory ending types. The effects of two other members of this family, BDNF and NT-4, on sensory neuron development are less clear. This study examined the role of brain-derived neurotrophic factor (BDNF) using transgenic mice that overexpress BDNF in epithelial target tissues of sensory neurons. BDNF transgenic mice had an increase in peripheral innervation density and showed selective effects on neuron survival. Neuron number in trigeminal ganglia, DRG, and SCG were unchanged, although a 38% increase in neurons comprising the placode-derived nodose-petrosal complex occurred. BDNF transgenic skin showed notable enhancement of innervation to hair follicles as detected by PGP9.5 immunolabeling. In nonhairy plantar skin, Meissner corpuscle sensory endings were larger, and the number of Merkel cells with associated innervation was increased. In trigeminal ganglia, neurons expressing trkB receptor were increased threefold, whereas trkA-positive neurons doubled. Analysis of trkB by Northern, reverse transcription-PCR, and Western assays indicated a modest increase in the expression of the T1 truncated receptor and preferential distribution to the periphery. These data indicate that skin-derived BDNF does not enhance survival of cutaneous sensory neurons, although it does promote neurite innervation of specific sites and sensory end organs of the skin.

Bacteriophage-bacteriophage interactions in the evolution of pathogenic bacteria.

Many bacteriophages carry virulence genes encoding proteins that play a major role in bacterial pathogenesis. Recently, investigators have identified bacteriophage-bacteriophage interactions in the bacterial host cell that also contribute significantly to the virulence of bacterial pathogens. The relationships between the bacteriophages pertain to one bacteriophage providing a helper function for another, unrelated bacteriophage in the host cell. Accordingly, these interactions can involve the mobilization of bacteriophage DNA by another bacteriophage, for example in Escherichia coli, Vibrio coli and Staphylococcus aureus; the host receptor for one bacteriophage being encoded by another, as found in V. cholerae; and the presence of one bacteriophage potentiating the virulence properties of another bacteriophage, as found in V. cholerae and Salmonella enterica.

Skin-derived nerve growth factor blocks programmed cell death in the trigeminal ganglia but does not enhance neuron proliferation.

Development of the cutaneous sensory nervous system is dependent on the production of neurotrophic factors, such as nerve growth factor (NGF), by the skin. Limited synthesis of NGF in developing skin is thought to underlie programmed cell death and cause a 50% neuronal loss. This loss does not occur in transgenic mice that overexpress NGF in the skin, which have double the number of neurons (J. Neurosci. 14 (1994) 1422). To determine whether increased NGF blocks neuronal death and/or increases neuronal precursor replication, we analyzed the trigeminal ganglia at embryonic days E12.5, E14.5 and E16.5 using transferase-mediated dUTP nick-end labeling (TUNEL) and bromodeoxyuridine labeling. Results show that excess target-derived NGF causes a major decrease in the percent of TUNEL-labeled neurons without affecting the percent of replicating neurons. Analysis of RNA and protein expression suggests this block in cell death is mediated via the anti-apoptotic protein bcl-2.

Basic and clinical aspects of visceral sensation: transmission in the CNS.

Pain and discomfort are the leading cause for consultative visits to gastroenterologists. Acute pain should be considered a symptom of an underlying disease, thereby serving a physiologically important function. However, many patients experience chronic pain in the absence of potentially harmful stimuli or disorders, turning pain into the primary problem rather than a symptom. Vagal and spinal afferents both contribute to the sensory component of the gut-brain axis. Current evidence suggests that they convey different elements of the complex sensory experience. Spinal afferents play a key role in the discriminatory dimension, while vagal input primarily affects the strong emotional and autonomic reactions to noxious visceral stimuli. Drugs, surgical and non-pharmacological treatments can target these pathways and provide therapeutic options for patients with chronic visceral pain syndromes.

Evaluating prolactin response to dopamine agonists in schizophrenia. Methodological problems.

Serum prolactin (PRL) level was assessed after challenges with apomorphine hydrochloride, saline, dopamine hydrochloride, or levodopa-carbidopa (Sinemet) in 19 control and 38 chronic schizophrenic subjects. Baseline PRL level varied inversely with age. High correlations existed between baseline PRL level and any subsequent absolute measure of PRL after administration of a dopamine agonist or placebo. Percent decrease was not a function of baseline concentrations and was therefore the only independent measure of drug response. Baseline PRL level was generally lower during exacerbation than remission in patients studied during two states of illness. Percent PRL level decrease after apomorphine administration was significantly greater in normal subjects than in schizophrenics. Correction of apomorphine responses for corresponding placebo (saline) values abolished differences between groups. Prolactin responses after dopamine or levodopa-carbidopa did not differ; however, placebo correction was not possible.

Apomorphine and schizophrenia. Treatment, CSF, and neuroendocrine responses.

Previous studies have variably reported the efficacy of apomorphine in treatment of schizophrenia and tardive dyskinesia. Stimulation of dopamine neuron autoreceptors is the presumed mode of action. Low-dose apomorphine (0.75 mg subcutaneously) and placebo were administered to 25 male schizophrenics to evaluate the drug's effect on psychotic and tardive dyskinetic symptoms. No significant improvement or deterioration was seen. Concomitant measurements of plasma prolactin and growth hormone levels and CSF homovanillic acid level indicated that the dose used was centrally active. These results indicate that an active though nonsedating dose of apomorphine does not ameliorate symptoms of schizophrenia or tardive dyskinesia.

Simultaneous protection of G156A methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and sensitization of tumor cells using O6-benzylguanine and temozolomide.

O6-Benzylguanine (BG) potentiates temozolomide (TMZ) cytotoxicity in tumors by inactivating O6-alkylguanine DNA alkyltransferase but also increases toxicity in hematopoietic cells. To improve the hematopoietic cell tolerance to alkylating agents, we retrovirally transduced the BG-resistant mutant G156A methylguanine DNA methyltransferase gene (deltaMGMT) into hematopoietic progenitors and evaluated whether deltaMGMT expression in hematopoietic colony-forming units would result in greater drug resistance to TMZ. DeltaMGMT expression in human and mouse colony-forming units followed by BG treatment resulted in a >7.7-fold increase in the TMZ 90% inhibitory concentration (IC90) and a 5.6-fold increase in the 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) IC90 relative to untransduced cells. This degree of protection enabled deltaMGMT-transduced CD34 cells to become much more resistant to BG and TMZ than SW480 cells, which express high O6-alkylguanine DNA alkyltransferase and are normally resistant to TMZ or BCNU alone. DeltaMGMT-transduced long-term culture initiating cells were also resistant to the BG and TMZ combination, as were untransduced long-term culture initiating cells, suggesting that noncycling early progenitors may be partially protected from TMZ. These data indicate that retroviral transduction of deltaMGMT into hematopoietic progenitors followed by BG and TMZ treatment may selectively protect hematopoietic cells more efficiently than BCNU, allowing dose-intensive and repetitive therapy without the risk of cumulative myelosuppression.