Effect of astaxanthin in type-2 diabetes -induced APPxhQC transgenic and NTG mice.

OBJECTIVES: Aggregation and misfolding of amyloid beta (Aß) and tau proteins, suggested to arise from post-translational modification processes, are thought to be the main cause of Alzheimer's disease (AD). Additionally, a plethora of evidence exists that links metabolic dysfunctions such as obesity, type 2 diabetes (T2D), and dyslipidemia to the pathogenesis of AD. We thus investigated the combinatory effect of T2D and human glutaminyl cyclase activity (pyroglutamylation), on the pathology of AD and whether astaxanthin (ASX) treatment ameliorates accompanying pathophysiological manifestations. METHODS: Male transgenic AD mice, APPxhQC, expressing human APP751 with the Swedish and the London mutation and human glutaminyl cyclase (hQC) enzyme and their non-transgenic (NTG) littermates were used. Both APPxhQC and NTG mice were allocated to 3 groups, control, T2D-control, and T2D-ASX. Mice were fed control or high fat diet ± ASX for 13 weeks starting at an age of 11-12 months. High fat diet fed mice were further treated with streptozocin for T2D induction. Effects of genotype, T2D induction, and ASX treatment were evaluated by analysing glycemic readouts, lipid concentration, Aß deposition, hippocampus-dependent cognitive function and nutrient sensing using immunosorbent assay, ELISA-based assays, western blotting, immunofluorescence staining, and behavioral testing via Morris water maze (MWM), respectively. RESULTS: APPxhQC mice presented a higher glucose sensitivity compared to NTG mice. T2D-induced brain dysfunction was more severe in NTG compared to the APPxhQC mice. T2D induction impaired memory functions while increasing hepatic LC3B, ABCA1, and p65 levels in NTG mice. T2D induction resulted in a progressive shift of Aß from the soluble to insoluble form in APPxhQC mice. ASX treatment reversed T2D-induced memory dysfunction in NTG mice and in parallel increased hepatic pAKT while decreasing p65 and increasing cerebral p-S6rp and p65 levels. ASX treatment reduced soluble Aß38 and Aß40 and insoluble Aß40 levels in T2D-induced APPxhQC mice. CONCLUSIONS: We demonstrate that T2D induction in APPxhQC mice poses additional risk for AD pathology as seen by increased Aß deposition. Although ASX treatment reduced Aß expression in T2D-induced APPxhQC mice and rescued T2D-induced memory impairment in NTG mice, ASX treatment alone may not be effective in cases of T2D comorbidity and AD.

Astaxanthin enhances autophagy, amyloid beta clearance and exerts anti-inflammatory effects in in vitro models of Alzheimer's disease-related blood brain barrier dysfunction and inflammation.

Defective degradation and clearance of amyloid-ß as well as inflammation per se are crucial players in the pathology of Alzheimer's disease (AD). A defective transport across the blood-brain barrier is causative for amyloid-ß (Aß) accumulation in the brain, provoking amyloid plaque formation. Using primary porcine brain capillary endothelial cells and murine organotypic hippocampal slice cultures as in vitro models of AD, we investigated the effects of the antioxidant astaxanthin (ASX) on Aß clearance and neuroinflammation. We report that ASX enhanced the clearance of misfolded proteins in primary porcine brain capillary endothelial cells by inducing autophagy and altered the Aß processing pathway. We observed a reduction in the expression levels of intracellular and secreted amyloid precursor protein/Aß accompanied by an increase in ABC transporters ABCA1, ABCG1 as well as low density lipoprotein receptor-related protein 1 mRNA levels. Furthermore, ASX treatment increased autophagic flux as evidenced by increased lipidation of LC3B-II as well as reduced protein expression of phosphorylated S6 ribosomal protein and mTOR. In LPS-stimulated brain slices, ASX exerted anti-inflammatory effects by reducing the secretion of inflammatory cytokines while shifting microglia polarization from M1 to M2 phenotype. Our data suggest ASX as potential therapeutic compound ameliorating AD-related blood brain barrier impairment and inflammation.

Liver X Receptor Activation Attenuates Oxysterol-Induced Inflammatory Responses in Fetoplacental Endothelial Cells.

Oxysterols are oxidized cholesterol derivatives whose systemic levels are found elevated in pregnancy disorders such as gestational diabetes mellitus (GDM). Oxysterols act through various cellular receptors and serve as a key metabolic signal, coordinating inflammation. GDM is a condition of low-grade chronic inflammation accompanied by altered inflammatory profiles in the mother, placenta and fetus. Higher levels of two oxysterols, namely 7-ketocholesterol (7-ketoC) and 7ß-hydroxycholesterol (7ß-OHC), were observed in fetoplacental endothelial cells (fpEC) and cord blood of GDM offspring. In this study, we tested the effects of 7-ketoC and 7ß-OHC on inflammation and investigated the underlying mechanisms involved. Primary fpEC in culture treated with 7-ketoC or 7ß-OHC, induced the activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signaling, which resulted in the expression of pro-inflammatory cytokines (IL-6, IL-8) and intercellular cell adhesion molecule-1 (ICAM-1). Liver-X receptor (LXR) activation is known to repress inflammation. Treatment with LXR synthetic agonist T0901317 dampened oxysterol-induced inflammatory responses. Probucol, an inhibitor of LXR target gene ATP-binding cassette transporter A-1 (ABCA-1), antagonized the protective effects of T0901317, suggesting a potential involvement of ABCA-1 in LXR-mediated repression of inflammatory signaling in fpEC. TLR-4 inhibitor Tak-242 attenuated pro-inflammatory signaling induced by oxysterols downstream of the TLR-4 inflammatory signaling cascade. Taken together, our findings suggest that 7-ketoC and 7ß-OHC contribute to placental inflammation through the activation of TLR-4. Pharmacologic activation of LXR in fpEC decelerates its shift to a pro-inflammatory phenotype in the presence of oxysterols.

miR-181a Modulation of ERK-MAPK Signaling Sustains DC-SIGN Expression and Limits Activation of Monocyte-Derived Dendritic Cells.

DC-SIGN+ monocyte-derived dendritic cells (mo-DCs) play important roles in bacterial infections and inflammatory diseases, but the factors regulating their differentiation and proinflammatory status remain poorly defined. Here, we identify a microRNA, miR-181a, and a molecular mechanism that simultaneously regulate the acquisition of DC-SIGN expression and the activation state of DC-SIGN+ mo-DCs. Specifically, we show that miR-181a promotes DC-SIGN expression during terminal mo-DC differentiation and limits its sensitivity and responsiveness to TLR triggering and CD40 ligation. Mechanistically, miR-181a sustains ERK-MAPK signaling in mo-DCs, thereby enabling the maintenance of high levels of DC-SIGN and a high activation threshold. Low miR-181a levels during mo-DC differentiation, induced by inflammatory signals, do not support the high phospho-ERK signal transduction required for DC-SIGNhi mo-DCs and lead to development of proinflammatory DC-SIGNlo/- mo-DCs. Collectively, our study demonstrates that high DC-SIGN expression levels and a high activation threshold in mo-DCs are linked and simultaneously maintained by miR-181a.

Amyloid-beta impairs insulin signaling by accelerating autophagy-lysosomal degradation of LRP-1 and IR-ß in blood-brain barrier endothelial cells in vitro and in 3XTg-AD mice.

Aberrant insulin signaling constitutes an early change in Alzheimer's disease (AD). Insulin receptors (IR) and low-density lipoprotein receptor-related protein-1 (LRP-1) are expressed in brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). There, insulin may regulate the function of LRP-1 in Aß clearance from the brain. Changes in IR-ß and LRP-1 and insulin signaling at the BBB in AD are not well understood. Herein, we identified a reduction in cerebral and cerebrovascular IR-ß levels in 9-month-old male and female 3XTg-AD (PS1M146V, APPSwe, and tauP301L) as compared to NTg mice, which is important in insulin mediated signaling responses. Reduced cerebral IR-ß levels corresponded to impaired insulin signaling and LRP-1 levels in brain. Reduced cerebral and cerebrovascular IR-ß and LRP-1 levels in 3XTg-AD mice correlated with elevated levels of autophagy marker LC3B. In both genotypes, high-fat diet (HFD) feeding decreased cerebral and hepatic LRP-1 expression and elevated cerebral Aß burden without affecting cerebrovascular LRP-1 and IR-ß levels. In vitro studies using primary porcine (p)BCEC revealed that Aß peptides 1-40 or 1-42 (240 nM) reduced cellular levels and interaction of LRP-1 and IR-ß thereby perturbing insulin-mediated signaling. Further mechanistic investigation revealed that Aß treatment accelerated the autophagy-lysosomal degradation of IR-ß and LRP-1 in pBCEC. LRP-1 silencing in pBCEC decreased IR-ß levels through post-translational pathways further deteriorating insulin-mediated responses at the BBB. Our findings indicate that LRP-1 proves important for insulin signaling at the BBB. Cerebral Aß burden in AD may accelerate LRP-1 and IR-ß degradation in BCEC thereby contributing to impaired cerebral and cerebromicrovascular insulin effects.

Astaxanthin exerts protective effects similar to bexarotene in Alzheimer's disease by modulating amyloid-beta and cholesterol homeostasis in blood-brain barrier endothelial cells.

The pathogenesis of Alzheimer's disease (AD) is characterized by overproduction, impaired clearance, and deposition of amyloid-ß peptides (Aß) and connected to cholesterol homeostasis. Since the blood-brain barrier (BBB) is involved in these processes, we investigated effects of the retinoid X receptor agonist, bexarotene (Bex), and the peroxisome proliferator-activated receptor α agonist and antioxidant, astaxanthin (Asx), on pathways of cellular cholesterol metabolism, amyloid precursor protein processing/Aß production and transfer at the BBB in vitro using primary porcine brain capillary endothelial cells (pBCEC), and in 3xTg AD mice. Asx/Bex downregulated transcription/activity of amyloidogenic BACE1 and reduced Aß oligomers and ~80 kDa intracellular 6E10-reactive APP/Aß species, while upregulating non-amyloidogenic ADAM10 and soluble (s)APPα production in pBCEC. Asx/Bex enhanced Aß clearance to the apical/plasma compartment of the in vitro BBB model. Asx/Bex increased expression levels of ABCA1, LRP1, and/or APOA-I. Asx/Bex promoted cholesterol efflux, partly via PPARα/RXR activation, while cholesterol biosynthesis/esterification was suppressed. Silencing of LRP-1 or inhibition of ABCA1 by probucol reversed Asx/Bex-mediated effects on levels of APP/Aß species in pBCEC. Murine (m)BCEC isolated from 3xTg AD mice treated with Bex revealed elevated expression of APOE and ABCA1. Asx/Bex reduced BACE1 and increased LRP-1 expression in mBCEC from 3xTg AD mice when compared to vehicle-treated or non-Tg treated mice. In parallel, Asx/Bex reduced levels of Aß oligomers in mBCEC and Aß species in brain soluble and insoluble fractions of 3xTg AD mice. Our results suggest that both agonists exert beneficial effects at the BBB by balancing cholesterol homeostasis and enhancing clearance of Aß from cerebrovascular endothelial cells.

Prazosin induced lysosomal tubulation interferes with cytokinesis and the endocytic sorting of the tumour antigen CD98hc.

The quinazoline based drug prazosin (PRZ) is a potent inducer of apoptosis in human cancer cells. We recently reported that PRZ enters cells via endocytosis and induces tubulation of the endolysosomal system. In a proteomics approach aimed at identifying potential membrane proteins with binding affinity to quinazolines, we detected the oncoprotein CD98hc. We confirmed shuttling of CD98hc towards lysosomes and upregulation of CD98hc expression in PRZ treated cells. Gene knockout (KO) experiments revealed that endocytosis of PRZ still occurs in the absence of CD98hc - suggesting that PRZ does not enter the cell via CD98hc but misroutes the protein towards tubular lysosomes. Lysosomal tubulation interfered with completion of cytokinesis and provoked endoreplication. CD98hc KO cells showed reduced endoreplication capacity and lower sensitivity towards PRZ induced apoptosis than wild type cells. Thus, loss of CD98hc does not affect endocytosis of PRZ and lysosomal tubulation, but the ability for endoreplication and survival of cells. Furthermore, we found that glutamine, lysomototropic agents - namely chloroquine and NH4Cl - as well as inhibition of v-ATPase, interfere with the intracellular transport of CD98hc. In summary, our study further emphasizes lysosomes as target organelles to inhibit proliferation and to induce cell death in cancer. Most importantly, we demonstrate for the first time that the intracellular trafficking of CD98hc can be modulated by small molecules. Since CD98hc is considered as a potential drug target in several types of human malignancies, our study possesses translational significance suggesting, that old drugs are able to act on a novel target.

Endoplasmic reticulum stress in bipolar disorder? - BiP and CHOP gene expression- and XBP1 splicing analysis in peripheral blood.

BACKGROUND: Endoplasmic Reticulum stress activates the Unfolded Protein Response, which is partially impaired in Bipolar Disorder (BD) according to previous in-vitro studies. Thus, BiP and CHOP gene expression and XBP1 splicing were analyzed in peripheral blood of study participants with BD and controls. METHODS: RNA was isolated from fasting blood of study participants with BD (n = 81) and controls (n = 54) and reverse transcribed into cDNA. BiP and CHOP gene expression was analyzed with quantitative RT-PCR. Atypical splicing of XBP1 mRNA was measured by semi-quantitative RT-PCR, gel-electrophoresis and densitometry. ANCOVAs with the covariates age, BMI, sex, lithium and anticonvulsants intake were used with SPSS. Bonferroni correction was used to correct for multiple testing (adjusted p = 0.0083). RESULTS: BiP gene expression was significantly higher in BD than in controls (F(1/128) = 10.076, p = 0.002, Partial η2 = 0.073). Total XBP1 (F(1/126) = 9.550, p = 0.002, Partial η2 = 0.070) and unspliced XBP1 (F(1/128)= 8.803, p= 0.004, Patial η2 = 0.065) were significantly decreased in BD. Spliced XBP1 (F(1/126) = 5.848, p = 0.017, Partial η2 = 0.044) and the ratio spliced XBP1/ unspliced XBP1 did not differ between BD and controls (F(1/126) = 0.599, p = 0.441, Partial η2 = 0.005). Gene expression did not differ between euthymia, depression and mania. DISCUSSION: BiP gene expression was significantly higher in BD compared to controls. Total and unspliced XBP1 were significantly lower in BD than in the control group. Thus, both genes may be considered as putative trait markers. Nevertheless, XBP1 splicing itself did not differ between both groups.

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-ß clearance in blood-brain barrier endothelial cells.

Amyloid-ß peptides (Aß) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aß metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aß. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aß metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aß oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aß clearance across the pBCEC monolayer. Treatment of pBCEC with Aß(1-40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3×Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aß oligomers and reduced Aß uptake and cell-associated Aß oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aß processing and clearance at the BBB.

Endoplasmic Reticulum Stress and Bipolar Disorder - Almost Forgotten Therapeutic Drug Targets in the Unfolded Protein Response Pathway Revisited.

Bipolar Disorder (BD) is characterized by recurring mood swings, which are not completely understood yet. So far, it is an accepted theory that multiple factors contribute to pathogenesis of BD according to the vulnerability-stressmodel. This model combines on the one hand biological predisposing vulnerability, and on the other hand several chronic and acute stressful negative events as underlying mechanisms of BD. Recently, ER (Endoplasmic Reticulum) stress reached the spotlight of BD research again. The expression of the chaperone BiP (syn. GRP78/glucose-regulated protein, 78kDa), which is highly expressed in the Endoplasmic Reticulum (ER), is upregulated by different kinds of mood stabilizers. These results implied that the ER, an organelle which is prone towards different kinds of cellular stress, might be involved in the pathophysiology of BD. This hypothesis was further strengthened by hypothesis driven genetic association studies, which showed significant association of BiP promotor polymorphisms with BD. Also other ER-stress associated genes like XBP1 (X-box binding protein 1) or GRP94 (glucose-regulated protein, 94kDa, synonym for heat shock protein HSP90B1) were recently linked to BD in hypothesis driven gene association studies. In addition to the proteins mentioned before, our review focuses on further UPR (Unfolded Protein Response) related proteins associated with BD and raises the hypothesis that ER-stress may represent a common interface between BD and obesity which is overrepresented in BD patients. Finally, members of the UPR pathway are discussed as putative targets for mood stabilizers.

The cytotoxicity of the α1-adrenoceptor antagonist prazosin is linked to an endocytotic mechanism equivalent to transport-P.

Since the α1-adrenergic antagonist prazosin (PRZ) was introduced into medicine as a treatment for hypertension and benign prostate hyperplasia, several studies have shown that PRZ induces apoptosis in various cell types and interferes with endocytotic trafficking. Because PRZ is also able to induce apoptosis in malignant cells, its cytotoxicity is a focus of interest in cancer research. Besides inducing apoptosis, PRZ was shown to serve as a substrate for an amine uptake mechanism originally discovered in neurones called transport-P. In line with our hypothesis that transport-P is an endocytotic mechanism also present in non-neuronal tissue and linked to the cytotoxicity of PRZ, we tested the uptake of QAPB, a fluorescent derivative of PRZ, in cancer cell lines in the presence of inhibitors of transport-P and endocytosis. Early endosomes and lysosomes were visualised by expression of RAB5-RFP and LAMP1-RFP, respectively; growth and viability of cells in the presence of PRZ and uptake inhibitors were also tested. Cancer cells showed co-localisation of QAPB with RAB5 and LAMP1 positive vesicles as well as tubulation of lysosomes. The uptake of QAPB was sensitive to transport-P inhibitors bafilomycin A1 (inhibits v-ATPase) and the antidepressant desipramine. Endocytosis inhibitors pitstop(®) 2 (general inhibitor of endocytosis), dynasore (dynamin inhibitor) and methyl-ß-cyclodextrin (cholesterol chelator) inhibited the uptake of QAPB. Bafilomycin A1 and methyl-ß-cyclodextrin but not desipramine were able to preserve growth and viability of cells in the presence of PRZ. In summary, we confirmed the hypothesis that the cellular uptake of QAPB/PRZ represents an endocytotic mechanism equivalent to transport-P. Endocytosis of QAPB/PRZ depends on a proton gradient, dynamin and cholesterol, and results in reorganisation of the LAMP1 positive endolysosomal system. Finally, the link seen between the cellular uptake of PRZ and cell death implies a still unknown pro-apoptotic membrane protein with affinity towards PRZ.

The anti-hypertensive drug prazosin induces apoptosis in the medullary thyroid carcinoma cell line TT.

BACKGROUND/AIM: Medullary thyroid carcinoma (MTC) is a tumor associated with poor prognosis since it exhibits high resistance against conventional cancer therapy. Recent studies have shown that quinazolines exhibit a pro-apoptotic effect on malignant cells. The aim of the present study was to elucidate whether MTC cells are affected by quinazolines, in particular prazosin. MATERIALS AND METHODS: Proliferation, apoptosis and cell morphology of the MTC cell line TT were analyzed by WST-1 assay, caspase 3/7 activation tests and microscopy. Fibroblasts were used as control for non-malignant cells. RESULTS: Prazosin potently inhibited the growth of TT cells, induced apoptosis and caused vacuolization, as well as needle-like filopodia. Fibroblasts were affected by prazosin in the same way as MTC cells. CONCLUSION: MTC cells are responsive to prazosin treatment similar to other malignancies. The fact that fibroblasts also respond to prazosin further highlights the importance to identify the unknown pro-apoptotic target of quinazolines.

Effect of posttranscriptional regulatory elements on transgene expression and virus production in the context of retrovirus vectors.

Ineffective transgene expression in a sufficient amount of target cells is still a limitation in retroviral vector mediated gene therapy. Thus, we systematically evaluated four genetic modulators, (i) the woodchuck posttranscriptional regulatory element (WPRE), (ii) the mouse RNA transport element (RTE), (iii) the constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), and (iv) the 5' untranslated region of the human heat shock protein 70 (Hsp70 5'UTR), all of them involved in the posttranscriptional control of mRNA nucleo/cytoplasmatic transport, RNA stability, and translation efficiency, in an MLV-based retrovirus vector context. Insertion of the WPRE into the retrovirus vector resulted in enhancement of transgene expression (EGFP) both in transfected virus producing cells as well as in infected recipient cells irrespective of the location in the vector. The best effect was observed with two copies of the WPRE, 3' of the transgene and in the 3' untranslated region of the vector backbone. However, oligomerization of this element does not further increase transgene expression. Presence of the WPRE resulted also in an increase in virus production. Introduction of the CTE and/or RTE in the retroviral vector did not alter transgene expression and infectious particle production. Positive effects were observed only in vectors harboring the CTE and/or RTE in combination with the WPRE. The activity of the Hsp70 5'UTR as a translational enhancer was found to be negligible in the context of the retroviral vector. However, interference of the Hsp70 5'UTR strong secondary structure with the packaging sequence of the viral RNA was experimentally excluded as being the cause of this. These data suggest that only the WPRE is a suitable element for the improvement of transgene expression and oncoretroviral vector production.

Effects of sequences of prokaryotic origin on titer and transgene expression in retroviral vectors.

Transcriptionally targeted MLV-based ProCon vectors allow expression of the transduced gene in a promoter-specific manner by replacement of the viral U3 region with a heterologous promoter. In order to evaluate the effects of sequence elements present in ProCon vectors on transgene expression (enhanced green fluorescence protein, EGFP), a series of deletion constructs mimicking the situation in proviral DNA following promoter conversion, where expression of the EGFP gene is driven by three different constitutive promoters (MLV U3, mCMV, and hCMV) in the context of a 5'LTR, respectively, were generated and tested in transient transfection experiments. We discovered that modifications in the 3'LTR have only marginal effects on the EGFP expression and the sequence between the promoter and the transgene did not influence EGFP expression at all. On the other hand, EGFP expression was reduced by up to 17-fold in cells transfected with constructs containing SV40neo and/or pBR322ori sequences. To study this effect in transduced cells, we generated a series of retroviral vectors in which these elements were deleted in various combinations and found that an increase in EGFP expression and viral titer was also consistently obtained using vectors lacking these elements, although this was much smaller than that observed using the expression constructs. A vector containing the gene for puromycin resistance (pac) in place of the neomycin resistance gene (neo) was also tested, and found to result in improved vector titers and transgene expression. We conclude that, where possible, the inclusion of neo and ori sequences in retroviral vectors should be avoided, and that, if selection of infected cells is necessary, the pac, rather than neo gene should be used.

Multiple modifications allow high-titer production of retroviral vectors carrying heterologous regulatory elements.

Tumor-specific expression of therapeutic genes is a prerequisite in many approaches to retrovirus-mediated cancer gene therapy. However, tissue specificity is often associated with a reduction in viral titer. To overcome this problem, we constructed a series of murine leukemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors carrying either the simian virus 40 poly(A) signal trimer (3pA) inserted in the 3' long terminal repeat (LTR) of these vectors or the human cytomegalovirus enhancer region (CMVe) inserted 5' and 3' of the retroviral LTRs. Furthermore, an extended AT stretch/attachment site (AT/att) of wild-type MLV was introduced into the vector. In the vector-producing cells, insertion of the CMVe and/or the 3pA resulted in a three- to fourfold-enhanced marker gene expression compared to the parental vector, whereas insertion of the AT/att gave a slight decrease in expression. The combination of all three modifications had no additional effects. In contrast, however, neomycin selection of infected cells revealed only a slight increase in virus titer with vectors carrying the 3pA modification; the titer was increased by 1 with vectors containing the extended AT/att, although the viral DNA copy numbers in infected cells were similar with both types of vectors. Thus, insufficient integration rather than insufficient reverse transcription and/or production of virus RNA is the major cause for the low titer obtained with the ProCon vectors. The combination of all three modifications resulted in a 2- to 3-log increase in the virus titer. These modifications result in expression targeted ProCon vectors with titers similar to those of nonmodified MLV-based vectors.