Is there a multidisciplinary role for 5-methyltetrahydrofolate? The obstetric evidence in perspective.

5-methyltetrahydrofolate (5-MTHF), or its synthetic precursor, folic acid, is traditionally used as a supplement for improving fertility and for the prevention of embryonal neural tube defects. However, in the last decade, starting from the effectiveness of this preventive treatment in the gynecological setting, the use of 5-MTHF was extended to other medical and pathological areas. Thus, there might be a rationale for the use of 5-MTHF for purposes other than the protection of the growing embryo linked to the possible effect of MTHFR variants in different pathological conditions. A narrative review was conducted to provide an overview of the available evidence on the use of 5-MTHF in the obstetric field and to critically discuss the available data regarding the use of 5-MTHF across other different therapeutic areas. Results showed that the use of 5-MTHF in pregnancy presents some advantages if compared with folic acid, such as immediate action, the non-necessity of metabolic activation, and the immediate bioavailability of the mother and fetus. Otherwise, the role of 5-MTHF in the management of cardiovascular risk is still debated due to the multiple confounding factors that characterize this patient setting. A link between folate deficiency in pregnancy and postpartum depression has been proposed, as well as between folate levels and the onset of depression. In conclusion, evidence from the literature supports the additional role of 5-MTHF as a pleiotropic drug with a transversal effect in different therapeutic contexts. With regard to the prevention of cardiovascular disorders, available evidence is not conclusive.

Vibrational spectroscopy to study ancient Roman funerary practices at the "Hypogeum of the Garlands" (Italy).

The "Hypogeum of the Garlands" is a sepulchral site, recently found in Grottaferrata (Lazio, Italy), dating back to the first-second century AD. Two sarcophagi were discovered inside, hosting the human remains of Aebutia Quarta, a rich Roman woman, and her son Carvilius Gemellus. While the body of Carvilius is exceptionally well-preserved, following its embalming and perfect sealing of the sarcophagus, in the case of Aebutia only the bones were preserved because of the sarcophagus's seal breaking down, although she was covered with perfectly preserved flower garlands. Embalming of the body was a rare ritual in the Imperial Roman times when corpses were more often cremated. The remains of Aebutia showed possible traces of heating. Burned bones from a third individual were discovered on the chamber's floor and preliminary anthropological survey showed that this individual was a male of 40-50 years old. Here, a combination of spectroscopic techniques, including non-destructive inelastic neutron scattering and Raman spectroscopy, and minimally destructive Fourier transform infrared spectroscopy, were applied to the analysis of these bone samples to give information about ancient Roman funerary practices. The temperature and burning conditions were thus determined, showing that Aebutia Quarta was exposed to mild temperatures (200 °C) only in the upper part of the body, while the third individual was likely cremated as its bones were exposed to temperatures up to 900 °C in quasi-anaerobic conditions.

Heterologous production of the widely used natural food colorant carminic acid in Aspergillus nidulans.

The natural red food colorants carmine (E120) and carminic acid are currently produced from scale insects. The access to raw material is limited and current production is sensitive to fluctuation in weather conditions. A cheaper and more stable supply is therefore desirable. Here we present the first proof-of-concept of heterologous microbial production of carminic acid in Aspergillus nidulans by developing a semi-natural biosynthetic pathway. Formation of the tricyclic core of carminic acid is achieved via a two-step process wherein a plant type III polyketide synthase (PKS) forms a non-reduced linear octaketide, which subsequently is folded into the desired flavokermesic acid anthrone (FKA) structure by a cyclase and a aromatase from a bacterial type II PKS system. The formed FKA is oxidized to flavokermesic acid and kermesic acid, catalyzed by endogenous A. nidulans monooxygenases, and further converted to dcII and carminic acid by the Dactylopius coccus C-glucosyltransferase DcUGT2. The establishment of a functional biosynthetic carminic acid pathway in A. nidulans serves as an important step towards industrial-scale production of carminic acid via liquid-state fermentation using a microbial cell factory.

Muscle fiber diameter assessment in cleft lip using image processing.

OBJECTIVE: To pilot investigation of muscle fiber diameter (MFD) on medial and lateral sides of the cleft in 18 infants with cleft lip with or without cleft palate (CL/P) using image processing. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded (FFPE) tissue samples from the medial and lateral sides of the cleft were analyzed for MFD using an image-processing program (ImageJ). For within-case comparison, a paired Student's t test was performed. For comparisons between classes, an unpaired t test was used. RESULTS: Image processing enabled rapid measurement of MFD with majority of fibers showing diameter between 6 and 11 µm. There was no significant difference in mean MFD between the medial and lateral sides, or between CL and CLP. However, we found a significant difference on the medial side (p = .032) between males and females. CONCLUSION: The image processing on FFPE tissues resulted in easy quantification of MFD with finding of a smaller MFD on the medial side in males suggesting possible differences in orbicularis oris (OO) muscle between the two sexes in CL that warrants replication using larger number of cases. Moreover, this finding can aid subclinical phenotyping and potentially in the restoration of the anatomy and function of the upper lip.

Tooth agenesis and orofacial clefting: genetic brothers in arms?

Tooth agenesis and orofacial clefts represent the most common developmental anomalies and their co-occurrence is often reported in patients as well in animal models. The aim of the present systematic review is to thoroughly investigate the current literature (PubMed, EMBASE) to identify the genes and genomic loci contributing to syndromic or non-syndromic co-occurrence of tooth agenesis and orofacial clefts, to gain insight into the molecular mechanisms underlying their dual involvement in the development of teeth and facial primordia. Altogether, 84 articles including phenotype and genotype description provided 9 genomic loci and 26 gene candidates underlying the co-occurrence of the two congenital defects: MSX1, PAX9, IRF6, TP63, KMT2D, KDM6A, SATB2, TBX22, TGFα, TGFß3, TGFßR1, TGFßR2, FGF8, FGFR1, KISS1R, WNT3, WNT5A, CDH1, CHD7, AXIN2, TWIST1, BCOR, OFD1, PTCH1, PITX2, and PVRL1. The molecular pathways, cellular functions, tissue-specific expression and disease association were investigated using publicly accessible databases (EntrezGene, UniProt, OMIM). The Gene Ontology terms of the biological processes mediated by the candidate genes were used to cluster them using the GOTermMapper (Lewis-Sigler Institute, Princeton University), speculating on six super-clusters: (a) anatomical development, (b) cell division, growth and motility, (c) cell metabolism and catabolism, (d) cell transport, (e) cell structure organization and (f) organ/system-specific processes. This review aims to increase the knowledge on the mechanisms underlying the co-occurrence of tooth agenesis and orofacial clefts, to pave the way for improving targeted (prenatal) molecular diagnosis and finally to reflect on therapeutic or ultimately preventive strategies for these disabling conditions in the future.

Insertion of the LINE-1 element in the C-MYC gene and immunoreactivity of C-MYC, p53, p21 and p27 proteins in different morphological patterns of the canine TVT / Inserção do elemento LINE - 1 no gene C-MYC e imunorreatividade das proteinas C-MYC, p53, p21 e p27 nos diferentes padrões morfológicos do tumor TVT

The canine transmissible venereal tumor (TVT) affects the external genitalia of dogs by the natural transplant of viable tumor cells. Thus, this research aimed to diagnose and characterize TVT morphological patterns, identify the insertion of the LINE-1 element in C-MYC gene, by means of the polymerase chain reaction (PCR), and evaluate the immunohistochemical expression of C-MYC, p53, p21 and p27 proteins. The relationship between C-MYC and p53 proteins and their interference on the expression of p21 and p27 were also studied. For that, 20 samples of naturally occurring TVT were used, subjected to cytopathological, histopathological and immunohistochemical analysis, and to molecular diagnosis of neoplasia. The increased tissue expression and the correlation among C-MYC, p53, p21 and p27 proteins indicate reduction and/or loss of their functionality in the TVT microenvironment, with consequent apoptotic suppression, maintenance of cell growth and progression of neoplasia.(AU)

O tumor venéreo transmissível canino (TVT) afeta a genitália externa de cães pelo transplante natural de células tumorais viáveis. Assim, esta pesquisa teve como objetivo diagnosticar e caracterizar TVT em padrões morfológicos, identificar a inserção do elemento LINE-1 em gene C-MYC, por meio da reação em cadeia da polimerase (PCR), e avaliar a expressão imuno-histoquímica do C-MYC, p53, p21 e p27. A relação entre C-MYC e as proteínas p53 e a sua interferência na expressão de p21 e p27 foram também estudadas. Para isso, foram utilizadas 20 amostras de ocorrência natural de TVT, submetido a exame citopatológico, histopatológica e imuno-histoquímica e ao diagnóstico molecular de neoplasia. A expressão aumentada do tecido e a correlação entre a C-MYC e as proteínas p53, p21 e p27 indicam redução e/ou perda de funcionalidade na TVT em seu microambiente, com consequente supressão apoptótica, manutenção do crescimento celular e progressão da neoplasia.(AU)

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites.

The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.

The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways.

The biosynthetic pathway for the cyanogenic glucoside dhurrin in sorghum has previously been shown to involve the sequential production of (E)- and (Z)-p-hydroxyphenylacetaldoxime. In this study we used microsomes prepared from wild-type and mutant sorghum or transiently transformed Nicotiana benthamiana to demonstrate that CYP79A1 catalyzes conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime whereas CYP71E1 catalyzes conversion of (E)-p-hydroxyphenylacetaldoxime into the corresponding geometrical Z-isomer as required for its dehydration into a nitrile, the next intermediate in cyanogenic glucoside synthesis. Glucosinolate biosynthesis is also initiated by the action of a CYP79 family enzyme, but the next enzyme involved belongs to the CYP83 family. We demonstrate that CYP83B1 from Arabidopsis thaliana cannot convert the (E)-p-hydroxyphenylacetaldoxime to the (Z)-isomer, which blocks the route towards cyanogenic glucoside synthesis. Instead CYP83B1 catalyzes the conversion of the (E)-p-hydroxyphenylacetaldoxime into an S-alkyl-thiohydroximate with retention of the configuration of the E-oxime intermediate in the final glucosinolate core structure. Numerous microbial plant pathogens are able to detoxify Z-oximes but not E-oximes. The CYP79-derived E-oximes may play an important role in plant defense.

Bone Changes in an Italian Ancient Human Skeleton--Possibly Caused by Leprosy.

Leprosy is an infectious disease caused by Mycobacterium leprae and Mycobacterium lepromatosis. In the last stage it can afflict the skeleton with a series of specific and non-specific bone changes. The possibility of studyingthe skeleton of an individual who lived in the pre-antibiotic era (Roman period) with skeletal changes in the rhino-maxillary region and hand and foot bones, permitted skeletal lesions to be analyzed directly. In addition, the localization and the complexity of the bony lesions could be attributed to the presence of leprosy. The importance of this approach was the possibility to verify the nature and typology of the primary and secondary bone changes in leprosy in absence of clinical therapy.

Strong association of variants around FOXE1 and orofacial clefting.

Nonsyndromic orofacial clefting (nsOFC) is a common, complex congenital disorder. The most frequent forms are nonsyndromic cleft lip with or without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO). Although they are generally considered distinct entities, a recent study has implicated a region around the FOXE1 gene in both nsCL/P and nsCPO. To investigate this hypothesis, we analyzed the 2 most strongly associated markers (rs3758249 and rs4460498) in 2 independent samples of differing ethnicities: Central European (949 nsCL/P cases, 155 nsCPO cases, 1163 controls) and Mayan Mesoamerican (156 nsCL/P cases, 10 nsCPO cases, 338 controls). While highly significant associations for both single-nucleotide polymorphisms were obtained in nsCL/P (rs4460498: p Europe = 6.50 × 10(-06), p Mayan = .0151; rs3758249: p Europe = 2.41 × 10(-05), p Mayan = .0299), no association was found in nsCPO (p > .05). Genotyping of rs4460498 in 472 independent European trios revealed significant associations for nsCL/P (p = .016) and nsCPO (p = .043). A meta-analysis of all data revealed a genomewide significant result for nsCL/P (p = 1.31 × 10(-08)), which became more significant when nsCPO cases were added (p nsOFC = 1.56 × 10(-09)). These results strongly support the FOXE1 locus as a risk factor for nsOFC. With the data of the initial study, there is now considerable evidence that this locus is the first conclusive risk factor shared between nsCL/P and nsCPO.

Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study.

A collection of 1,108 case-parent trios ascertained through an isolated, nonsyndromic cleft lip with or without cleft palate (CL/P) was used to replicate the findings from a genome-wide association study (GWAS) conducted by Beaty et al. (Nat Genet 42:525-529, 2010), where four different genes/regions were identified as influencing risk to CL/P. Tagging SNPs for 33 different genes were genotyped (1,269 SNPs). All four of the genes originally identified as showing genome-wide significance (IRF6, ABCA4 and MAF, plus the 8q24 region) were confirmed in this independent sample of trios (who were primarily of European and Southeast Asian ancestry). In addition, eight genes classified as 'second tier' hits in the original study (PAX7, THADA, COL8A1/FILIP1L, DCAF4L2, GADD45G, NTN1, RBFOX3 and FOXE1) showed evidence of linkage and association in this replication sample. Meta-analysis between the original GWAS trios and these replication trios showed PAX7, COL8A1/FILIP1L and NTN1 achieved genome-wide significance. Tests for gene-environment interaction between these 33 genes and maternal smoking found evidence for interaction with two additional genes: GRID2 and ELAVL2 among European mothers (who had a higher rate of smoking than Asian mothers). Formal tests for gene-gene interaction (epistasis) failed to show evidence of statistical interaction in any simple fashion. This study confirms that many different genes influence risk to CL/P.

Comparison of stem cell properties of cells isolated from normal and inflamed dental pulps.

AIM: To compare cells from normal and inflamed human dental pulps regarding the presence of stem cells, their proliferation and differentiation potential. METHODOLOGY: Human dental pulp stem cells (hDPSCs) were isolated from normal (DPSC-N) and inflamed dental pulps (DPSC-I). They were compared in respect to proliferation (MTT assay), morphology and STRO-1 expression. STRO-1-positive cells were subject to proliferation (MTT and CFU counting) and morphological analyses and then submitted to odonto-osteogenic, adipogenic and condrogenic differentiation. Differentiated cells were evaluated concerning morphology and the expression, by qRT-PCR, of BSP, LPL and SOX-9 genes. The amount of mineralized matrix produced after odonto-osteogenic differentiation was compared with quantitative Alizarin Red staining. RESULTS: No difference was observed in the morphology and in the proliferation rate of DPSC-N and DPSC-I either before or after separation of STRO-1-positive cells. These cells represented 0.46% (±0.14) and 0.43% (±0.19) of the cell population from normal and inflamed dental pulps, respectively. Both DPSC-N and DPSC-I were capable of differentiating under the three assayed conditions and presented similar patterns for BSP, LPL and SOX-9 expression. Mineralized matrix production was also compatible. In all the quantitative experiments, differences were found between cells from each patient, either from normal or from inflamed pulps. Nonetheless, there was no statistical difference between these two groups. CONCLUSION: The morphology, proliferation rate and differentiation potential of DPSC-I were similar to the observed in DPSC-N, thus demonstrating that the inflammatory process did not affect the stem cell properties that were assessed.

Cloning, characterization and heterologous expression of the first Penicillium echinulatum cellulase gene.

AIMS: Penicillium echinulatum is effective for bioconversion processes. However, nothing is known about the molecular biology of its cellulolytic system. We describe for the first time the isolation, cloning and expression of a P. echinulatum cellulase cDNA (Pe-egl1) encoding a putative endoglucanase. METHODS AND RESULTS: Pe-egl1 cDNA was identified from random sequencing of a P. echinulatum cDNA library. The deduced EGL1 protein possibly belongs to the glycosyl hydrolase family 5A, with 387 amino acid residues and strong similarity with other fungal endoglucanases. The cDNA was heterologously expressed in Pichia pastoris. The recombinant EGL1 secreted by a Pic. pastoris recombinant strain revealed the characteristics of particular interest: an optimal activity over a broad pH range (5.0-9.0), and an optimal temperature of 60 degrees C. The recombinant EGL1 also showed high thermostability (84% of residual activity after 1 h of pre-incubation at 70 degrees C). Calcium exerted a strong stimulatory effect over EGL1 activity. CONCLUSIONS: Altogether, these results point to the potential application of this P. echinulatum endoglucanase in cellulose processing industries, particularly the textile one because of its biochemical properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization and heterologous expression of the first P. echinulatun cDNA inaugurates the exploitation of this potential industrial micro-organism.

P2X7 gene polymorphisms do not appear to be a susceptibility gene locus in sporadic cases of systemic lupus erythematosus.

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. A loss-of-function single nucleotide polymorphism (SNP) at position 1513 (1513 A-->C) of the P2X(7) gene has recently been identified in both healthy and chronic lymphocytic leukemia (CLL) B-cells, translating into a loss of P2X(7)-mediated apoptosis in these cells. This antiapoptotic effect results in increased B-cell numbers, thereby potentially contributing to the survival of B-CLL clones. It was hypothesized that prolonged cell survival may also predispose to induction of autoimmunity. The objective of this study is to analyze the role of the P2X(7) receptor and its loss-of-function 1513 A-->C polymorphism (SNP) in the development of systemic lupus erythematosus (SLE). DNA samples obtained from patients with sporadic SLE were analyzed for the presence of the 1513 A-->C polymorphism using polymerase chain reaction (PCR) amplification and then direct sequencing. No significant difference in allele frequencies (1513 A-->C polymorphism) between sporadic cases of SLE and controls was found. A loss-of-function SNP at position 1513 (1513 A-->C) of the P2X(7) gene does not appear to be a susceptibility gene locus for the development of sporadic SLE.

HLA-G genotype and HLA-G expression in systemic lupus erythematosus: HLA-G as a putative susceptibility gene in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease mainly mediated by the deposit of immune complexes and defects in T lymphocytes and antigen-presenting cells along with a high production of T-helper 2 cytokines. A tolerance-inducible function of nonclassical class Ib human leukocyte antigen (HLA)-G molecule in innate and adaptive cellular responses has been reported, suggesting a role in inflammatory diseases. A 14 bp sequence insertion/deletion polymorphism (rs16375) in the 3'-untranslated region of the HLA-G gene has been associated to the stability of HLA-G messenger RNA. The insertion of the 14 bp sequence seems to be associated with lower levels of soluble HLA-G (sHLA-G). The aim of this study was to evaluate the possible association of the presence of the 14 bp sequence (+14 bp) with SLE. We have HLA-G genotyped 200 SLE patients and 451 healthy control subjects (HS; Italian) and analyzed the plasma levels of sHLA-G and interleukin-10 (IL-10) in a subset of SLE patients and healthy subjects (Italian and Danish). A significant increase of the +14 bp HLA-G allele was detected in the Italian SLE patients compared with HS [P = 0.003, OR 1.44 (95% CI 1.13-1.82)]. A significant increased frequency of HLA-G +14/+14 bp and a decreased frequency of HLA-G -14/-14 bp were observed in SLE patients. There median concentration of sHLA-G was significantly lower in the plasma of SLE patients compared with that in the plasma of healthy controls (P < 0.0001). Furthermore, the results confirmed higher concentrations of IL-10-positive plasma in SLE patients. These results support a potential role for HLA-G in the susceptibility of SLE.

Colorectal cancer screening: results of a 5-year program in asymptomatic subjects at increased risk.

BACKGROUND AND AIMS: The province of Ferrara has one of the highest incidences of colorectal cancer (CRC) in Italy. In January 2000, we set up a colonoscopy screening program focussing on first-degree relatives of CRC patients. We now report the results 5 years after the beginning of the project. SCREENEES AND METHODS: In October 1999, we started a campaign stressing the usefulness of colonoscopy for the first-degree relatives of CRC patients. Subjects included in the screening program were aged between 45 and 75 years with at least one first-degree relative affected by CRC. They were invited to an interview where a physician suggested colonoscopy as a screening option. RESULTS: In 5 years, 776 subjects were interviewed and 733 (94.4%) agreed to an endoscopic examination (M/F:375/401; mean age 55 years): 562 colonoscopies were performed. Adenomas and cancers were found in 122 (21.7%) and 12 (2.1%) subjects, respectively. Histological examination in 181 persons with lesions (32.8%) showed (most serious lesion quoted) 47 hyperplastic polyps (26% of all lesions), 2 serrated adenomas (1.1%), 68 tubular adenomas (48%), 24 tubulovillous adenomas (13.3%), 9 adenomas with high grade dysplasia (5%) and 12 adenocarcinomas (6.6%). The majority of the cancers were at an early stage (8 Dukes A and 3 Dukes B). Sedation was used in only 42 colonoscopies (7.5%). CONCLUSIONS: A colonoscopy-based screening in this selected high-risk population is feasible. Even without sedation subjects readily agreed to the endoscopic procedure. We identified a significant number of advanced neoplasms and cancers at an early stage suggesting that this could be a useful tool in early identification of CRC.

Development of strategies for the use of anti-growth factor treatments.

Aberrant signalling through the epidermal growth factor receptor (EGFR) is associated with increased cancer cell proliferation, reduced apoptosis, invasion and angiogenesis. Over-expression of the EGFR is seen in a variety of tumours and is a rational target for antitumour strategies. Among the classes of agent targeting the EGFR are small-molecule inhibitors, which include gefitinib (IRESSA), which acts by preventing EGFR phosphorylation and downstream signal transduction. De novo and acquired resistance, however, have been reported to gefitinib and here we describe evidence which indicates that the type II receptor tyrosine kinases (RTKs) insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (InsR) play important roles in the mediation of responses to gefitinib in the de novo- and acquired-resistance phenotypes in several cancer types. Moreover, combination strategies that additionally target the IGF-IR/InsR can enhance the antitumour effects of gefitinib.

Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer.

Breast cancer models of acquired tamoxifen resistance, oestrogen receptor (ER)+ /ER- de novo resistance and gene transfer studies cumulatively demonstrate the increased importance of growth factor receptor signalling, notably the epidermal growth factor receptor (EGFR)/HER2, in tamoxifen resistance. Our recent in vitro studies also suggest that EGFR signalling productively cross-talks with insulin-like growth factor receptor (IGF-1R) and, where present, activates ER on key AF-1 serine residues to facilitate acquired tamoxifen-resistant growth. This paper presents our immunohistochemical evidence that EGFR/HER2 signalling (i.e. transforming growth factor (TGF)alpha, EGFR and HER2 expression; phosphorylation of EGFR, HER2 and ERK1/2 MAP kinase) is also prominent in clinical de novo resistant and modestly increased in acquired tamoxifen-resistant states, suggesting that anti-EGFR/HER2 strategies may prove valuable treatments. Primary breast cancer samples employed were obtained for (1) patients subsequently treated with tamoxifen for advanced disease where endocrine response and survival data were available and (2) ER+ elderly patients during tamoxifen response and relapse. We also present our clinical immunohistochemical findings that IGF-1R expression, its phosphorylation on tyrosine 1316, and also phosphorylation on serine 118 of ER are not only prominent in ER+ tamoxifen-responsive disease, but are also detectable in ER+ de novo and acquired tamoxifen-resistant breast cancer, where there is evidence of EGFR/ER cross-talk. Our data suggest that agents to deplete effectively ER or IGF-1R signalling may be of value in treating ER+ de novo/acquired tamoxifen resistance in addition to tamoxifen-responsive disease in vivo. IGF-1R inhibitors may also prove valuable in ER- patients, since considerable IGF-1R signalling activity was apparent within approximately 50% of such tumours.

Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells.

De novo and acquired resistance to the anti-tumour drug gefitinib (ZD1839; Iressa), a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has been reported. We have determined whether signalling through the IGF-I receptor (IGF-1R) pathway plays a role in the gefitinib-acquired resistance phenotype. Continuous exposure of EGFR-positive MCF-7-derived tamoxifen resistant breast cancer cells (TAM-R) to 1 microM gefitinib resulted in a sustained growth inhibition (90%) for 4 months before the surviving cells resumed proliferation. A stable gefitinib-resistant subline (TAM/TKI-R) was established after a further 2 months and this showed no detectable basal phosphorylated EGFR activity. Compared with the parental TAM-R cells, the TAM/ TKI-R cells demonstrated (a) elevated levels of activated IGF-1R, AKT and protein kinase C (PKC)delta, (b) an increased sensitivity to growth inhibition by the IGF-1R TKI AG1024 and (c) an increased migratory capacity that was reduced by AG1024 treatment. Similarly, the EGFR-positive androgen-independent human prostate cancer cell line DU145 was also continuously challenged with 1 microM gefitinib and, although substantial growth inhibition (60%) was seen initially, a gefitinib-resistant variant (DU145/TKI-R) developed after 3 months. Like their breast cancer counterparts, the DU145/TKI-R cells showed increases in the levels of components of the IGF-1R signalling pathway and an elevated sensitivity to growth inhibition by AG1024 compared with the parent DU145 cell line. Additionally, DU145/TKI-R cell migration was also decreased by this inhibitor. We have therefore concluded that in breast and prostate cancer cells acquired resistance to gefitinib is associated with increased signalling via the IGF-1R pathway, which also plays a role in the invasive capacity of the gefitinib-resistant phenotype.