DHEA inhibits acute microglia-mediated inflammation through activation of the TrkA-Akt1/2-CREB-Jmjd3 pathway.

Dehydroepiandrosterone (DHEA) is the most abundant circulating steroid hormone in humans, produced by the adrenals, the gonads and the brain. DHEA was previously shown to bind to the nerve growth factor receptor, tropomyosin-related kinase A (TrkA), and to thereby exert neuroprotective effects. Here we show that DHEA reduces microglia-mediated inflammation in an acute lipopolysaccharide-induced neuro-inflammation model in mice and in cultured microglia in vitro. DHEA regulates microglial inflammatory responses through phosphorylation of TrkA and subsequent activation of a pathway involving Akt1/Akt2 and cAMP response element-binding protein. The latter induces the expression of the histone 3 lysine 27 (H3K27) demethylase Jumonji d3 (Jmjd3), which thereby controls the expression of inflammation-related genes and microglial polarization. Together, our data indicate that DHEA-activated TrkA signaling is a potent regulator of microglia-mediated inflammation in a Jmjd3-dependent manner, thereby providing the platform for potential future therapeutic interventions in neuro-inflammatory pathologies.

Pharmacokinetics and efficacy of intraocular flurbiprofen.

PURPOSE: Intravitreal delivery of non-steroidal anti-inflammatory drugs could be an effective way to treat macular edema caused by posterior segment inflammation. In this study, we evaluated the intravitreal bioavailability and anti-inflammatory efficacy of flurbiprofen in rabbit eyes. METHODS: For pharmacokinetics, 0.1 ml of 7.66 mg/ml flurbiprofen solution was injected intravitreally and vitreous drug levels were analyzed at specific time points using LC-MS technique. For efficacy, 100 ng lipopolysaccharide of E.coli was injected intravitreally in rabbits to induce inflammation. The animals were separated in three groups and received intraocular flurbiprofen, dexamethasone and PBS to serve as control. Complete ocular examination and total cell count in aqueous fluid were determined to evaluate the extent of inflammation. Eyes were then enucleated for histopathology analysis. The efficacy in the uveitis model was determined by clinical signs of inflammation, total leukocyte count and histology findings. RESULTS: No adverse events were observed during pharmacokinetic assessment. No signs of inflammation, hemorrhage or retina detachment were detected. The recovery of flurbiprofen from vitreous samples was 92.6%. The half-life of flurbiprofen was estimated to be 1.92 h with an elimination constant rate (K) of 0.36. Treatment with intraocular injections of flurbiprofen and dexamethasone significantly reduced total leukocyte count in a manner comparable to dexamethasone [reduction of 96.84% (p < 0.05) and 97.44% (p < 0.05), respectively]. Histologic studies demonstrated significantly less signs of ocular inflammation after flurbiprofen injection compared to control eyes. CONCLUSIONS: Flurbiprofen is effective in suppressing inflammation in this experimental uveitis model. In our experimental setting, intravitreal flurbiprofen seem to have a therapeutic result comparable to dexamethasone. However, the half-life of the drug remains short, necessitating further research to prolong its presence in the vitreous cavity.

Predictive and prognostic vaue of LPS-stimulated cytokine secretion in metastatic non-small cell lung cancer

OBJECTIVE: Cancer patients usually develop malnutrition which may alter their innate immune system integrity. The aim of this study was to investigate the clinical relevance of chemokine response after lipopolysaccharide (LPS)-stimulation in metastatic non-small cell lung cancer (NSCLC). METHODS: Blood samples from metastatic NSCLC patients were incubated with LPS before the onset of systemic therapy. Interleukin (IL)-6 and IL-8 levels at baseline and after LPS-stimulation were measured and the fold change compared to baseline levels was evaluated as the stimulation index for each cytokine per patient. Results were correlated with sex, age, smoking status, histologic subtype, performance status (PS), albumin, Mini Nutritional Assessment (MNA) status and clinical outcomes. RESULTS: Totally 103 patients were evaluated. Mean (±SD) stimulation index was 37.6 (±57.8) for IL-6 and 76.7 (±133.4) for IL-8. The disease control rate after first-line chemotherapy was 44/80 (55 %) and the mean (±SD) progression-free survival (PFS) and overall survival (OS) were 4.2 (±3.9) and 9.2 (±1.1) months, respectively. MNA, PS, albumin, IL-6 and IL-8 stimulation indices were univariately associated with PFS and OS. IL-8 stimulation index emerged as an independent predictor of both PFS and OS, along with PS, and albumin levels. CONCLUSION: The extent of IL-6 and IL-8 stimulation after ex vivo induction with LPS is an important predictor of clinical outcome in metastatic NSCLC patients (AU)

Predictive and prognostic value of LPS-stimulated cytokine secretion in metastatic non-small cell lung cancer.

OBJECTIVE: Cancer patients usually develop malnutrition which may alter their innate immune system integrity. The aim of this study was to investigate the clinical relevance of chemokine response after lipopolysaccharide (LPS)-stimulation in metastatic non-small cell lung cancer (NSCLC). METHODS: Blood samples from metastatic NSCLC patients were incubated with LPS before the onset of systemic therapy. Interleukin (IL)-6 and IL-8 levels at baseline and after LPS-stimulation were measured and the fold change compared to baseline levels was evaluated as the stimulation index for each cytokine per patient. Results were correlated with sex, age, smoking status, histologic subtype, performance status (PS), albumin, Mini Nutritional Assessment (MNA) status and clinical outcomes. RESULTS: Totally 103 patients were evaluated. Mean (±SD) stimulation index was 37.6 (±57.8) for IL-6 and 76.7 (±133.4) for IL-8. The disease control rate after first-line chemotherapy was 44/80 (55 %) and the mean (±SD) progression-free survival (PFS) and overall survival (OS) were 4.2 (±3.9) and 9.2 (±1.1) months, respectively. MNA, PS, albumin, IL-6 and IL-8 stimulation indices were univariately associated with PFS and OS. IL-8 stimulation index emerged as an independent predictor of both PFS and OS, along with PS, and albumin levels. CONCLUSION: The extent of IL-6 and IL-8 stimulation after ex vivo induction with LPS is an important predictor of clinical outcome in metastatic NSCLC patients.

Treatment of experimental murine colitis with CD40 antisense oligonucleotides delivered in amphoteric liposomes.

BACKGROUND AND AIMS: CD40-CD40L interactions appear to play an important role in the pathogenesis of experimental colitis. We tested the effect and investigated the underlying mechanism of action of systemically administered antisense oligonucleotide (ASO) targeting CD40 formulated in amphoteric liposomes (nov038/CD40). The charge characteristics of the amphoteric liposomes (anionic surface charge at physiological pH that becomes cationic at low pH), facilitate efficient sequestration of the ASO inside the liposomes at low pH and the direction of the carriers towards macrophages and dendritic cells under physiological conditions. METHODS: Colitis was induced in Balb/c mice using 2,4,6-Trinitrobenzene sulphonic acid (TNBS) and treated with nov038/CD40. Disease was monitored by body weight, histology, cytokine profiling and changes in immune cell populations. CD40 expression on different cell subsets was analyzed by flow cytometry. An antigen challenge model was used to determine neoimmunity under CD40 modulation. RESULTS: Administration of nov038/CD40 inhibited the development of TNBS colitis as assessed by weight loss, histology and cytokine profiles; unformulated CD40 ASO or nov038 encapsulating an unrelated ASO (nov038/SCR) were ineffective. The novel agent is potent as it completely suppressed even established colitis with a single treatment and significantly reduced T-cell activation as well as levels of pro-inflammatory mediators in serum. The inhibition of CD40 specifically occurred in macrophages, but not in B-cells. In contrast to prednisolone, standard treatment for inflammatory bowel diseases (IBD) that is effective in a single administration and involves extensive immunosuppression, nov038/CD40 did not affect the number of B- or Treg cells. Eventually, we observed a largely intact neoimmunity under conditions of a CD40 inhibition. CONCLUSIONS: Administration of nov038/CD40, but neither naked CD40 ASO nor nov038/SCR, prevents the development and treats established colitis in mice. Delivery of CD40 ASO in nov038 is highly cell-specific as it selectively suppresses CD40 on macrophages, but not on B-cells; the novel agent has strong anti-inflammatory characteristics without being immunosuppressive.

Aberrant expression of corticotropin-releasing hormone in pre-eclampsia induces expression of FasL in maternal macrophages and extravillous trophoblast apoptosis.

Corticotropin-releasing hormone (CRH) and its receptors are expressed in human placenta. Recently, the impaired function of this system has been associated with a number of complications of pregnancy, including pre-eclampsia. The aim of the study was to test the hypothesis that CRH participates in the pathophysiology of pre-eclampsia through the induction of macrophage-mediated apoptosis of extravillous trophoblasts (EVTs). We found that the expression of CRH was increased in the EVT of the placental bed biopsy specimens from pre-eclamptic pregnancies (1.8-fold increase; P < 0.05). In addition, significantly larger numbers of apoptotic EVT were detected in pre-eclamptic placentas compared with normal ones (P < 0.05), and only in pre-eclamptic placentas, decidual macrophages were found to be Fas ligand (FasL)-positive. In vitro studies on the effect of CRH on human macrophages suggested that CRH induced the expression of the FasL protein in human macrophages and potentiated their ability to induce the apoptosis of a Fas-expressing EVT-based hybridoma cell line in co-cultures. These findings demonstrate a possible mechanism by which the aberrant expression of CRH in pre-eclampsia may activate the FasL-positive decidual macrophages, impair the physiological turnover of EVT and eventually disturb placentation.

Correlation between serum 25-hydroxyvitamin D levels and quadriceps muscle strength in elderly cretans.

Vitamin D deficiency is associated with osteomalacic myopathy, and muscle weakness related to vitamin D deficiency has been implicated as a possible risk factor for falls in the elderly. This study investigated the possible correlation between serum 25-hydroxyvitamin D (25[OH]D) concentration and quadriceps muscle strength in ambulatory community dwelling Cretan men (n=13) and women (n=35) aged > or = 65 years. Quadriceps muscle strength was measured isometrically using Cybex 6000 apparatus. The mean serum 25(OH)D concentration was significantly higher in men than in women (76.00 versus 49.11 nmol/l, respectively). Serum 25(OH)D values were < 50 nmol/l in 15% of men and in 60% of women. Serum 25(OH)D concentration correlated positively with quadriceps muscle strength. In conclusion, vitamin D deficiency was common in the study participants despite the high levels of sunlight in Crete. Serum 25(OH)D levels were positively correlated with muscle strength.

The metabolic syndrome in early pregnancy and risk of gestational diabetes mellitus.

AIM: The objective of the present study was to determine whether or not maternal metabolic syndrome in early pregnancy in women without previous diabetes is associated with the development of gestational diabetes mellitus (GDM). METHODS: A total of 508 women from the Rhea study-involving a pregnant cohort in Crete, Greece (2007-2009)-with singleton pregnancies were included in the present analysis. Maternal fasting serum samples were collected and blood pressure measured before gestational week 15. The metabolic syndrome in early pregnancy was defined according to NHLBI/AHA criteria. Pregnant women were screened for GDM between weeks 24 and 28 of gestation, as defined by Carpenter and Coustan criteria. Multivariable log-binomial regression models were used to estimate the effect of the metabolic syndrome in early pregnancy on the risk of GDM, after adjusting for confounding factors. RESULTS: Women with the metabolic syndrome were at high risk of GDM (RR=3.17; 95% CI: 1.06-9.50). Among the components of the metabolic syndrome, the most significant risk factors were impaired fasting glucose (RR=4.92; 95% CI: 1.41-17.23) and pre-pregnancy obesity (RR=2.65; 95% CI: 1.23-5.70). A 10-mmHg rise in systolic and diastolic blood pressure increased the relative risk of GDM by 49% (RR=1.49; 95% CI: 1.10-2.02) and 34% (RR=1.34; 95% CI: 1.04-1.73), respectively, whereas a 1-unit increase in pre-pregnancy BMI increased the relative risk of GDM by 6% (RR=1.06; 95% CI: 1.01-1.12). CONCLUSION: These findings suggest that women with the metabolic syndrome in early pregnancy have a greater risk of developing GDM.

The corticotropin-releasing factor (CRF) family of peptides as local modulators of adrenal function.

Corticotropin-releasing factor (CRF), also termed corticotropin-releasing hormone (CRH) or corticoliberin, is the major regulator of the adaptive response to internal or external stresses. An essential component of the adaptation mechanism is the adrenal gland. CRF regulates adrenal function indirectly through the central nervous system (CNS) via the hypothalamic-pituitary-adrenal (HPA) axis and via the autonomic nervous system by way of locus coeruleus (LC) in the brain stem. Accumulating evidence suggests that CRF and its related peptides also affect the adrenals directly, i.e. not through the CNS but from within the adrenal gland where they form paracrine regulatory loops. Indeed, CRF and its related peptides, the urocortins (UCNs: UCN1, UCN2 and UCN3), their receptors CRF type 1 (CRF(1)) and 2 (CRF(2)) as well as the endogenous pseudo-receptor CRF-binding protein (CRF-BP) are all expressed in adrenal cortical, medullary chromaffin and resident immune cells. The intra-adrenal CRF-based regulatory system is complex and depends on the balance between the local concentration of CRF ligands and the availability of their receptors.

Corticotropin-releasing factor (CRF) and the urocortins differentially regulate catecholamine secretion in human and rat adrenals, in a CRF receptor type-specific manner.

Corticotropin-releasing factor (CRF) affects catecholamine production both centrally and peripherally. The aim of the present work was to examine the presence of CRF, its related peptides, and their receptors in the medulla of human and rat adrenals and their direct effect on catecholamine synthesis and secretion. CRF, urocortin I (UCN1), urocortin II (UCN2), and CRF receptor type 1 (CRF1) and 2 (CRF2) were present in human and rat adrenal medulla as well as the PC12 pheochromocytoma cells by immunocytochemistry, immunofluorescence, and RT-PCR. Exposure of dispersed human and rat adrenal chromaffin cells to CRF1 receptor agonists induced catecholamine secretion in a dose-dependent manner, an effect peaking at 30 min, whereas CRF2 receptor agonists suppressed catecholamine secretion. The respective effects were blocked by CRF1 and CRF2 antagonists. CRF peptides affected catecholamine secretion via changes of subplasmaliminal actin filament polymerization. CRF peptides also affected catecholamine synthesis. In rat chromaffin and PC12 cells, CRF1 and CRF2 agonists induced catecholamine synthesis via tyrosine hydroxylase. However, in human chromaffin cells, activation of CRF1 receptors induced tyrosine hydroxylase, whereas activation of CRF2 suppressed it. In conclusion, it appears that a complex intraadrenal CRF-UCN/CRF-receptor system exists in both human and rat adrenals controlling catecholamine secretion and synthesis.

Dehydroepiandrosterone sulfate and allopregnanolone directly stimulate catecholamine production via induction of tyrosine hydroxylase and secretion by affecting actin polymerization.

Adrenal cortical cells of zona reticularis produce the neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester dehydroepiandrosterone sulfate (DHEAS), and allopregnanolone (ALLO). An interaction between zona reticularis and adrenal medulla has been postulated based on their close proximity and their interwoven borders. The aim of this paper was to examine in vitro the possible paracrine effects of these steroids on catecholamine production from adrenomedullary chromaffin cells, using an established in vitro model of chromaffin cells, the PC12 rat pheochromocytoma cell line. We have found the following: 1) DHEA, DHEAS, and ALLO increased acutely (peak effect between 10-30 min) and dose-dependently (EC50 in the nanomolar range) catecholamine levels (norepinephrine and dopamine). 2) It appears that the acute effect of these steroids involved actin depolymerization/actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. Specifically, 10(-6) m phallacidin, an actin filament stabilizer, completely prevented steroid-induced catecholamine secretion. 3) DHEAS and ALLO, but not DHEA, also affected catecholamine synthesis. Indeed, DHEAS and ALLO increased catecholamine levels at 24 h, an effect blocked by L-2-methyl-3-(-4-hydroxyphenyl)alanine and 3-(hydrazinomethyl)phenol hydrochloride, inhibitors of tyrosine hydroxylase and L-aromatic amino acid decarboxylase, respectively, suggesting that this effect involved catecholamine synthesis. The latter hypothesis was confirmed by finding that DHEAS and ALLO increased both the mRNA and protein levels of tyrosine hydroxylase. In conclusion, our findings suggest that neuroactive steroids exert a direct tonic effect on adrenal catecholamine synthesis and secretion. These data associate the adrenomedullary malfunction observed in old age and neuroactive steroids.

Urocortin in human gastric mucosa: relationship to inflammatory activity.

The presence of CRH and urocortin (Ucn), members of the CRH family of neuropeptides, was examined in human gastric biopsies from normal controls and in patients with active gastritis from Helicobacter pylori (H. pylori) and after eradication treatment. RT-PCR analysis showed the presence of the Ucn transcript in biopsies (obtained by gastroscopy) from normal and inflamed gastric mucosa, whereas the CRH transcript was not detectable. Immunoreactive (ir-) Ucn was localized (by immunohistochemistry) in gastric epithelial cells and in inflammatory elements of the surrounding negative for Ucn gastric stroma. The level of ir-Ucn was higher in gastric biopsies from the group of patients with active H. pylori gastritis than in normal controls (10.4 +/- 1.8 vs. 2.0 +/- 1.3 pg/ micro g total protein; P < 0.001). After the apparent eradication of H. pylori infection (by clinical and morphological criteria) ir-Ucn levels increased dramatically to 43.1 +/- 9.8 pg/ micro g total protein, (P < 0.001) compared with pretreatment values. Interestingly, nonresponders to the eradication treatment did not show any significant change in ir-Ucn levels (18.7 +/- 12.3 pg/ micro g total protein) compared with their pretreatment values. In conclusion, our data suggest that in human gastric epithelium Ucn is present and plays an important physiological role, whereas CRH is absent. In addition, and in contrast to what has been found for CRH in ulcerative colitis, a highly significant, but negative, correlation has been found between Ucn levels and gastric inflammation, suggesting that Ucn may exert an antiinflammatory effect in gastric mucosa.

Normal ras genes: their onco-suppressor and pro-apoptotic functions (review).

The ras family of oncogenes has been extensively studied for its implication in several types of human malignancies. Activation of ras genes involves mutations that alter the catalytic activity of the protein enhancing the downstream signals mostly towards cell proliferation and malignant transformation. Ras genes are also involved in induction of senescence or apoptosis, suggesting activation of alternative pathways that may be anti-oncogenic. Early experiments showed that transfection of wild-type ras in transformed cells reversed the oncogenic phenotype suggesting that wild-type ras has onco-suppressive properties. Indeed, expression of wild-type ras genes in several human malignancies is associated with good prognosis. In tumors carrying mutant ras genes the levels of expression of the wild-type allele never exceeded the mutant counterpart, indicating that the wild-type protein suppresses the effect of the mutant one. Recent development of the Kras2 deficient mice provided the tool to study the role of wild-type ras genes in tumorigenesis.

Tpl-2 induces apoptosis by promoting the assembly of protein complexes that contain caspase-9, the adapter protein Tvl-1, and procaspase-3.

The Tpl-2 proto-oncoprotein promotes cellular proliferation when overexpressed in a variety of tumor cell lines. Here, we present evidence that when overexpressed in immortalized non-transformed cells, Tpl-2 induces apoptosis by promoting the activation of caspase-3 via a caspase-9-dependent mechanism, and that apoptosis is enhanced when Tpl-2 is co-expressed with the newly identified ankyrin repeat protein Tvl-1. The activation of caspase-3 by caspase-9 is known to depend on the assembly of a multimolecular complex that includes Apaf-1 and caspase-9. Data presented here show that co-expression of Tpl-2 with Tvl-1 promotes the assembly of a complex that involves several proteins that bind Apaf-1 including Tvl-1, itself, Tpl-2 and phosphorylated procaspase-9. More important, procaspase-3, which under normal growth conditions is not associated with the complex, binds Tvl-1 conditionally in response to Tpl-2-generated apoptotic signals. The conditional association of procaspase-3 with Tvl-1 promotes the in vivo proteolytic maturation of procaspase-3 by caspase-9, a process casually linked to apoptosis.

The role of oncogenic kinases in human cancer (Review).

Tumorigenesis in humans is a multistep process, which reflects genetic alterations that lead to cell transformation and malignancy. Cellular genes that are altered are normally involved in maintaining cell homeostasis by participating in signaling pathways tightly regulated to maintain the functional integrity of the cell. When these genes are altered they escape from the regulatory control and transmit signals that lead to the progressive conversion of normal cells into cancer cells. Oncogenic signals involve activation of kinases, which can be either a primary event when they are directly mutated in a tumor cell or a secondary event as recipients and mediators of oncogenic signals. Transmembrane (e.g. EGFR, PDGFR) or cytoplasmic (Src, Abl) tyrosine kinases are found mutated in a variety of human tumors. Cytoplasmic serine threonine kinases (Raf, Akt, Tpl-2) are also mutated or activated in several types of human malignancies. Kinases transduce signals that lead to cell proliferation or inhibition of programmed cell death by activating transcription factors (e.g. AP1, NFkappaB, Myc), inhibiting pro-apoptotic molecules (e.g. Bad, Bax), or they participate in deregulating the cell cycle control. Thus, kinases play a central role in oncogenesis rendering them putative targets for anti-cancer drug design.

TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway.

Tpl2 knockout mice produce low levels of TNF-alpha when exposed to lipopolysaccharide (LPS) and they are resistant to LPS/D-Galactosamine-induced pathology. LPS stimulation of peritoneal macrophages from these mice did not activate MEK1, ERK1, and ERK2 but did activate JNK, p38 MAPK, and NF-kappaB. The block in ERK1 and ERK2 activation was causally linked to the defect in TNF-alpha induction by experiments showing that normal murine macrophages treated with the MEK inhibitor PD98059 exhibit a similar defect. Deletion of the AU-rich motif in the TNF-alpha mRNA minimized the effect of Tpl2 inactivation on the induction of TNF-alpha. Subcellular fractionation of LPS-stimulated macrophages revealed that LPS signals transduced by Tpl2 specifically promote the transport of TNF-alpha mRNA from the nucleus to the cytoplasm.

Mechanisms of retrovirus-induced oncogenesis.

Retroviruses are implicated in a series of human and animal tumours such as leukaemias, mammary tumours or skin cancer. The mechanism that they use to induce tumour formation varies. Insertional mutagenesis is a common mechanism in rodent, feline and avian retroviruses, where the retrovirus integrates into the host genome and affects the transcription of the neighbouring genes. Cloning of these affected genes led to identification of a series of oncogenes that play a significant role in the induction of human neoplasms. Retrovirus insertion also serves as a model to identify collaborating oncogenes. Human retroviruses use different, more complex mechanisms contributing to oncogenesis. Studies of the propagation and induction mechanisms used by retroviruses have given insight to the understanding of oncogenesis.

Overexpression of the Tpl-2/Cot oncogene in human breast cancer.

Tpl-2/Cot proto-oncogene encodes a serine threonine kinase and was initially cloned as a provirus insertion site in MoMuLV-induced T cell lymphomas in rats. Tpl-2 locus was also shown to be affected by provirus insertion in MMTV-induced mammary carcinomas in mice. The involvement of Tpl-2 in 35 human breast paired tumour specimens versus their corresponding adjacent normal tissue was evaluated. Tpl-2 was found overexpressed in 14 of the 35 breast tumours tested using a semi-quantitative RT - PCR method. Gene amplification was detected in eight out of the 14 specimens overexpressing Tpl-2, suggesting the increased number of copies of Tpl-2 gene as a possible mechanism for Tpl-2 overexpression. Significant association was found between the overexpression of Tpl-2 and stage I of the tumours, indicating that this molecular alteration may be an early event in the development of the disease. Furthermore, overexpression of Tpl-2 was associated with positive progesterone receptor status of the samples. This is the first report on the Tpl-2 oncogene linked to human breast tumours suggesting that it may be a key molecule for the study of human breast cancer.

The fit-1 common integration locus in human and mouse is closely linked to MYB.

The fit-1 locus was originally identified as a common insertion site for feline leukemia virus (FeLV) in thymic lymphosarcomas induced by FeLV-myc recombinant viruses, suggesting that it harbors a gene that cooperates with Myc in T-cell leukemogenesis. We have previously mapped the fit-1 locus to feline Chromosome (Chr) B2. We have now identified conserved sequences that allow the mapping of the murine homolog using the European Interspecific Backcross (EUCIB). This shows that fit-1 is located on mouse Chr 10, 1cM proximal to Ahi-1, a murine retroviral integration locus that is closely linked to Myb. Moreover, the physical linkage to MYB is maintained in the human genome, as shown by cloning of the human homolog of fit-1 from a Chr 6 cosmid library and a series of overlapping PAC clones. Generation of a contig map around the human homolog of fit-1 reveals that it is approximately 100-kb upstream of MYB. In addition to fit-1 and Ahi-1, two other common insertion sites, Mis-2 and Mml-1, have also been mapped adjacent to Myb on mouse Chr 10. Previous analysis of tumors carrying insertions at fit-1, Mml-1, Mis-2 and Ahi-1 showed no obvious abnormalities in Myb expression. However, the cluster of viral insertion loci in this region suggests either the presence of a closely linked activation target or that subtle effects on Myb have been overlooked.