Variable loss of functional activities of androgen receptor mutants in patients with androgen insensitivity syndrome.

Androgen receptor (AR) mutations in androgen insensitivity syndrome (AIS) are associated with a variety of clinical phenotypes. The aim of the present study was to compare the molecular properties and potential pathogenic nature of 8 novel and 3 recurrent AR variants with a broad variety of functional assays. Eleven AR variants (p.Cys177Gly, p.Arg609Met, p.Asp691del, p.Leu701Phe, p.Leu723Phe, p.Ser741Tyr, p.Ala766Ser, p.Arg775Leu, p.Phe814Cys, p.Lys913X, p.Ile915Thr) were analyzed for hormone binding, transcriptional activation, cofactor binding, translocation to the nucleus, nuclear dynamics, and structural conformation. Ligand-binding domain variants with low to intermediate transcriptional activation displayed aberrant Kd values for hormone binding and decreased nuclear translocation. Transcriptional activation data, FxxFF-like peptide binding and DNA binding correlated well for all variants, except for p.Arg609Met, p.Leu723Phe and p.Arg775Leu, which displayed a relatively higher peptide binding activity. Variants p.Cys177Gly, p.Asp691del, p.Ala766Ser, p.Phe814Cys, and p.Ile915Thr had intermediate or wild type values in all assays and showed a predominantly nuclear localization in living cells. All transcriptionally inactive variants (p.Arg609Met, p.Leu701Phe, p.Ser741Tyr, p.Arg775Leu, p.Lys913X) were unable to bind to DNA and were associated with complete AIS. Three variants (p.Asp691del, p.Arg775Leu, p.Ile915Thr) still displayed significant functional activities in in vitro assays, although the clinical phenotype was associated with complete AIS. The data show that molecular phenotyping based on 5 different functional assays matched in most (70%) but not all cases.

Functional analysis of novel androgen receptor mutations in a unique cohort of Indonesian patients with a disorder of sex development.

Mutations in the androgen receptor (AR) gene, rendering the AR protein partially or completely inactive, cause androgen insensitivity syndrome, which is a form of a 46,XY disorder of sex development (DSD). We present 3 novel AR variants found in a cohort of Indonesian DSD patients: p.I603N, p.P671S, and p.Q738R. The aim of this study was to determine the possible pathogenic nature of these newly found unclassified variants. To investigate the effect of these variants on AR function, we studied their impact on transcription activation, AR ligand-binding domain interaction with an FxxLF motif containing peptide, AR subcellular localization, and AR nuclear dynamics and DNA-binding. AR-I603N had completely lost its transcriptional activity due to disturbed DNA-binding capacity and did not show the 114-kDa hyperphosphorylated AR protein band normally detectable after hormone binding. The patient with AR-I603N displays a partial androgen insensitivity syndrome phenotype, which is explained by somatic mosaicism. A strongly reduced transcriptional activity was observed for AR-Q738R, together with diminished interaction with an FxxLF motif containing peptide. AR-P671S also showed reduced transactivation ability, but no change in DNA- or FxxLF-binding capacity and interferes with transcriptional activity for as yet unclear reasons.

Detailed functional studies on androgen receptor mild mutations demonstrate their association with male infertility.

CONTEXT: Mutations in the androgen receptor (AR) gene can cause the androgen insensitivity syndrome (AIS). For complete and severe partial AIS, well-characterized in vitro functional assays can be used for genotype-phenotype correlation; however, for mild forms of AIS, as associated with male infertility, experimental evidence is scarce or lacking. In particular, optimal in vitro functional tests informative about the genotype-phenotype relation have not been described. OBJECTIVE: The objective of this study was to investigate the association among genotype and phenotype for AR mutations found in infertile males by conventional functional assays and additional in-depth studies performed with several gene reporters. DESIGN: To this aim, we selected four AR missense mutations associated with isolated male infertility (L547F and two novel mutations A474V and S650G) or partial AIS (Y571H). After introduction of the specific mutations in AR expression plasmid, we performed classical in vitro studies (Western immunoblotting, electrophoretic mobility shift assay, hormone-response curves) and transactivation assays with different reporter constructs (MMTV, Sc-ARU-TK, TAT-GRE- 2X, Slp-ARU-TK and PEM). RESULTS AND CONCLUSIONS: Our results showed that standard functional tests provide sufficient information only for severe AR mutations, whereas for AR mutations found in mild AIS patients with male infertility, only an extensive analysis with different in vitro systems, and in particular with PEM promoter, can give information on the functionality of the AR and therefore on the pathogenicity of the mutations and on genotype-phenotype correlation.

Gene expression profiling in human endometrial cancer tissue samples: utility and diagnostic value.

OBJECTIVE: Recently, gene expression profiling techniques have been used on several human cancers to classify tumor subgroups with a specific biological behavior, which were previously undetected by the conventional histopathologic staging systems. In the current study, the clinical usefulness and prognostic value of gene expression profiling in human endometrial carcinomas were studied. METHODS: A macro cDNA array, containing cDNAs of 588 genes selected from different areas of cancer research, was used to generate gene expression profiles of tumor tissue samples. The gene expression profiles of 12 endometrial cancers, 3 benign (e.g. noncancer) endometrial tissue samples and 3 myometrial tissue samples, taken from human surgical specimen, were compared. RESULTS: The efficacy to generate a gene expression profile of these tissue samples was 77%. The RNA samples could be randomly taken from the tissue samples and were highly reproducible. Cluster analysis of gene expression profiles of the different samples showed that the benign endometrial and the myometrial samples clustered separately from the tumor samples, indicating that the gene expression profiles were tissue specific and not patient specific. Cluster analysis of the tumor samples revealed two distinct tumor clusters. Ranking of the tumors in the two clusters showed high similarity with the histopathologic classification [International Federation of Gynecology and Obstetrics (FIGO) grading]. CONCLUSION: Classification of endometrial tumors on basis of their gene expression profiles showed similarity with the FIGO grading system.

Expression and copy number analysis of TRPS1, EIF3S3 and MYC genes in breast and prostate cancer.

The long arm of chromosome 8 is one of the most common regions of amplification in cancers of several organs, especially carcinomas of the breast and prostate. TRPS1, MYC and EIF3S3 genes are located in one of the minimal regions of amplification, 8q23-q24, and have been suggested to be the target genes of the amplification. Here, our goal was to study copy number and expression of the three genes in order to investigate the significance of the genes in breast and prostate cancer. By using fluorescence in situ hybridisation (FISH), we first found that TRPS1 and EIF3S3 were amplified together in about one-third of hormone-refractory prostate carcinomas. Next, we analysed the mRNA expression of the three genes by real-time quantitative RT-PCR and the gene copy number by FISH in six breast and five prostate cancer cell lines. Breast cancer cell line, SK-Br-3, which contained the highest copy number of all three genes, showed overexpression of only EIF3S3. Finally, the expression levels of TRPS1, EIF3S3 and MYC were measured in freshly frozen clinical samples of benign prostate hyperplasia (BPH), as well as untreated and hormone-refractory prostate carcinoma. The TRPS1 and MYC expression levels were similar in all prostate tumour groups, whereas EIF3S3 expression was higher (P=0.029) in prostate carcinomas compared to BPH. The data suggest that the expression of EIF3S3 is increased in prostate cancer, and that one of the mechanisms underlying the overexpression is the amplification of the gene.

Gender differences in expression of androgen receptor in tibial growth plate and metaphyseal bone of the rat.

In this study, we investigate the expression of the androgen receptor (AR) in the tibial growth plate and metaphyseal bone of male and female rats at the mRNA and protein level. Using in situ hybridization and immunohistochemistry, AR mRNA and protein were demonstrated in proliferating and early hypertrophic chondrocytes in the growth plate of 1-, 4-, and 7-week-old male and female rats. Immunostaining for AR was observed both in the nucleus and the cytoplasm. After sexual maturation at 12 and 16 weeks of age, AR expression decreased in both genders and was confined to a small rim of prehypertrophic chondrocytes. In female rats of 40 weeks of age, this expression pattern was still visible. In most age groups there was a tendency toward an increased AR mRNA expression in male vs. female rats except in the 7-week-old animals. At the protein level, sexually maturing 7-week-old male rats demonstrated a higher staining intensity compared to their female counterparts. At this stage, AR staining in the males was mainly confined to the nucleus, whereas in females staining was predominantly found in the cytoplasm. In the tibial metaphysis, AR mRNA was detected in lining cells, osteoblasts, osteocytes, and osteoclasts at all stages of development. At the protein level, a similar expression pattern was observed, except for an absence of immunostaining in the lining cells. The staining was both nuclear and cytoplasmic. In most age groups, mRNA and protein signals were higher in males compared with females. We have demonstrated the presence of AR mRNA and protein in the tibial growth plate and the underlying metaphyseal bone during development of the rat. In male rats, the presence of higher messenger and protein staining intensities, as well as preferential nuclear staining during sexual maturation, suggests that direct actions of androgens in chondrocytes and in bone forming cells may be involved in establishing the gender differences in the skeleton.

Structure and function of GC79/TRPS1, a novel androgen-repressible apoptosis gene.

Expression of death-signaling genes induces many biochemical cascades resulting in elimination of cells via apoptosis or programmed cell death. GC79/TRPS1 is a novel apoptosis associated gene that encodes a multitype zinc finger GATA-type transcription factor. Expression of GC79/TRPS1 is repressed in the rat ventral prostate and significantly elevated after androgen withdrawal by castration. Castration leads to regression of the prostate caused by apoptosis of androgen-dependent prostate cells. Prostate cancer consists of androgen-dependent and androgen-independent cells. The androgen-independent cells, usually present in the prostate of advanced prostate cancer patients do not have the ability to undergo apoptosis after androgen withdrawal. GC79/TRPS1 expression in androgen-dependent prostate cancer cells is repressed by androgens, while GC79/TRPS1 expression is hardly detectable in androgen-independent prostate cancer cells under cell culture conditions. This suggests that lack of GC79/TRPS1 expression could be a mechanism for the inability to induce the apoptotic pathway in androgen-independent prostate cancer cells after androgen withdrawal. This review will focus on the current knowlegde of the structure and function of GC79/TRPS1, a novel androgen-repressible apoptosis gene.

A novel gene on human chromosome 2p24 is differentially expressed between androgen-dependent and androgen-independent prostate cancer cells.

Identification of genes involved in the transition from androgen-dependent to androgen-independent prostate cancer is important to extend our current knowledge of the disease. Using differential display RT-PCR analysis between androgen-dependent and androgen-independent prostate cancer cells, we have identified a novel gene, designated GC109. GC109 harbours a putative Cys-His cluster, a nuclear localisation signal, a leucine zipper and a ret finger protein (rfp)-like domain. GC109 mRNA expression in normal human tissues was found not to be restricted to the prostate. However, using a variety of 15 human cancer cell lines, GC109 mRNA was preferentially expressed in androgen-dependent LNCaP-FGC, compared with androgen-independent LNCaP-LNO, DU145 and PC3 human prostate cancer cells. Finally, the GC109 gene was mapped on human chromosome 2p24. Based on its protein domain structure and chromosomal localisation, we hypothesise that GC109 may be involved in chromosomal rearrangements in prostate cancer.

Lessons to be learned from the androgen receptor.

Androgen action is mediated by the androgen receptor, a ligand dependent transcription factor, belonging to the superfamily of nuclear receptors. The two most important androgens are testosterone and 5a-dihydrotestosterone and their tissue specific actions are mediated by the same androgen receptor protein. Binding of androgens by the androgen receptor results in two consecutive conformational changes, which are different from those induced by anti-androgens. The androgen receptor can use different transactivation domains (AF1 and AF5, respectively, in the NH2-terminal domain and AF2-AD in the COOH-terminal domain) depending on the "form" of the receptor protein. The AF2 function is strongly dependent on the presence of nuclear receptor coactivators. Two AF functions are ligand dependent (AF-1 and AF2), whereas AF5 operates in a ligand independent way. The ligand dependency of AF-1 in the full length androgen receptor and the switch to AF5 in the COOH-terminal truncated androgen receptor strongly suggests a functional inhibitory action of the ligand-binding domain on AF-1 in the absence of ligand and on AF5 in the presence of ligand. In vivo experiments favour a ligand dependent functional interaction between the AF-2 AD core region in the ligand-binding domain with the NH2-terminal domain. This interaction might be either direct or indirect, requiring additional factors, and results in androgen receptor driven transcription activation. The androgen receptor protein can undergo two post-translational modifications during receptor activation. Firstly, upon synthesis the protein is rapidly phosphorylated to acquire hormone binding capacities and secondly, upon hormone binding an additional phosphorylation occurs during transformation to the DNA-binding transcription activation form.

Molecular basis of androgen insensitivity.

Androgens are important steroid hormones for expression of the male phenotype. They have characteristic roles during male sexual differentiation, during development and maintenance of secondary male characteristics, and during the initiation and maintenance of spermatogenesis. The two most important androgens in this respect are testosterone and 5 alpha-dihydrotestosterone. Each androgen has its own specific role during male sexual differentiation, testosterone is involved in the development and differentiation of Wolffian duct derived structures, whereas 5 alpha-dihydrotestosterone, a metabolite of testosterone, is the active ligand in the urogenital sinus and tubercle and their derived structures. The actions of androgens are mediated by the androgen receptor. This ligand dependent transcription factor belongs to the superfamily of nuclear receptors, including those for the other steroid hormones. The androgen receptor gene is located on the X-chromosome at Xq11--12 and codes for a protein with a molecular mass of approximately 110 kDa. Only one androgen receptor cDNA has been identified sofar, despite two different ligands. It is generally accepted that defects in the androgen receptor gene prevent the normal development of both internal and external male structures in 46, XY individuals. The end-organ resistance to androgens has been designated as androgen insensitivity syndrome (AIS) and is distinct from other forms of male pseudohermaphroditism like 17 beta-hydroxy-steroid dehydrogenase type 3 deficiency, leydig cell hypoplasia due to inactivating LH receptor mutations or 5 alpha-reductase type 2 deficiency. Furthermore, two additional pathological situations are associated with abnormal androgen receptor structure and function -- spinal and bulbar muscular atrophy (SBMA, or Kennedy's disease) and prostate cancer. In the AR gene, four different types of mutations have been detected in DNA from individuals with AIS -- (i) single point mutations resulting in amino acid substitutions or premature stopcodons; (ii) nucleotide insertions or deletions most often leading to a frame shift and premature termination; (iii) complete or partial gene deletions; and (iv) intronic mutations in either splice donor or acceptor sites, which affect the splicing of AR RNA. The main phenotypic characteristics of individuals with the complete androgen insensitivity syndrome (CAIS) are, female external genitalia, a short, blind ending vagina, the absence of Wolffian duct derived structures, the absence of a prostate, development of gynecomastia and the absence of pubic and axillary hair. Usually testosterone levels are elevated at the time of puberty, while also elevated LH levels are found. In the partial androgen insensitivity syndrome (PAIS) several different phenotypes are evident, ranging from individuals with predominantly a female appearance to persons with ambiguous genitalia, or individuals with a predominantly male phenotype. At puberty, elevated LH, testosterone and estradiol levels are observed. Individuals with mild symptoms of undervirilization (mild androgen insensitivity syndrome (MAIS)) and infertility have been described as well. Phenotypic variation between individuals in different families has been described for several mutations. However, in cases of CAIS no phenotypic variation has been described within one single family, in contrast to families with individuals with PAIS. In general AIS, can be routinely analyzed and more than 150 different mutations have been reported now. Differential diagnosis of AIS is possible with syndromes presenting with almost similar phenotypes but with a completely different molecular cause.

Expression in hematological malignancies of a glucocorticoid receptor splice variant that augments glucocorticoid receptor-mediated effects in transfected cells.

Glucocorticoids play an important role in the treatment of a number of hematological malignancies, such as multiple myeloma. The effects of glucocorticoids are mediated through the glucocorticoid receptor alpha, the abundance of which can be modulated by alternative splicing of the glucocorticoid receptor mRNA. Two splice variants of the glucocorticoid receptor mRNA have been described: glucocorticoid receptor beta, which reportedly has a dominant negative effect on the actions of the glucocorticoid receptor alpha, and glucocorticoid receptor P, of which the effects are unknown. In this study, we have investigated the expression levels of these two splice variants at the mRNA level in multiple myeloma cells and in a number of other hematological tumors. Although the glucocorticoid receptor beta mRNA was, if at all, expressed at very low levels, considerable amounts (up to 50% of the total glucocorticoid receptor mRNA) glucocorticoid receptor P mRNA was present in most hematological malignancies. In transient transfection studies in several cell types and in multiple myeloma cell lines, the glucocorticoid receptor P increased the activity of the glucocorticoid receptor alpha. These results suggest that the relative levels of the glucocorticoid receptor alpha and the glucocorticoid receptor P may play a role in the occurrence of glucocorticoid resistance in tumor cells during the treatment of hematological malignancies with glucocorticoids.

Etiological studies of severe or familial hypospadias.

PURPOSE: Hypospadias is a congenital anomaly occurring in 1250 to 1830 live male births, of which 20% involve a severe type. The recurrence risk in families is high. In the majority of cases the underlying etiology remains unknown, which hampers further management based on the specific requirements associated with a specific etiology. MATERIALS AND METHODS: In a single center study 63 unselected cases of severe hypospadias were studied for all presently known causes of hypospadias using clinical as well as molecular biological techniques. Also, 16 families with hypospadias were analyzed for possible androgen receptor gene mutations. RESULTS: In 31% of cases of severe hypospadias the underlying etiology was identified. Of these 31% of cases 17% were due to complex genetic syndromes, 9.5% were due to chromosomal anomalies, and 1 involved the vanishing testes syndrome, the androgen insensitivity syndrome and 5alpha-reductase type 2 deficiency, respectively. Based on hormone stimulation tests Leydig cell hypoplasia and disorders of testosterone biosynthesis were suspected in some patients but not confirmed by mutation analysis of the respective genes. Familial hypospadias was due to androgen insensitivity in only 1 family but no other etiologies were identified in this group. CONCLUSIONS: Using patient history, physical examination, karyotyping, hormonal evaluation, including human chorionic gonadotropin testing in prepubertal cases and additional biochemical and molecular genetic evaluation, an etiological diagnosis was made in 31% of cases of severe hypospadias. This diagnosis has implications for further patient treatment. In addition, familial hypospadias is rarely due to the androgen insensitivity syndrome.

Phenotypic variation in a family with partial androgen insensitivity syndrome explained by differences in 5alpha dihydrotestosterone availability.

Mutations in the androgen receptor (AR) gene result in a wide range of phenotypes of the androgen insensitivity syndrome (AIS). Inter- and intrafamilial differences in the phenotypic expression of identical AR mutations are known, suggesting modifying factors in establishing the phenotype. Two 46,XY siblings with partial AIS sharing the same AR gene mutation, R846H, but showing very different phenotypes are studied. Their parents are first cousins. One sibling with grade 5 AIS was raised as a girl; the other sibling with grade 3 AIS was raised as a boy. In both siblings serum levels of hormones were measured; a sex hormone-binding globulin (SHBG) suppression test was completed; and mutation analysis of the AR gene, Scatchard, and SDS-PAGE analysis of the AR protein was performed. Furthermore, 5alpha-reductase 2 expression and activity in genital skin fibroblasts were investigated, and the 5alpha-reductase 2 gene was sequenced. The decrease in SHBG serum levels in a SHBG suppression test did not suggest differences in androgen sensitivity as the cause of the phenotypic variation. Also, androgen binding characteristics of the AR, AR expression levels, and the phosphorylation pattern of the AR on hormone binding were identical in both siblings. However, 5alpha-reductase 2 activity was normal in genital skin fibroblasts from the phenotypic male patient but undetectable in genital skin fibroblasts from the phenotypic female patient. The lack of 5alpha-reductase 2 activity was due to absent or reduced expression of 5alpha-reductase 2 in genital skin fibroblasts from the phenotypic female patient. Exon and flanking intron sequences of the 5alpha-reductase 2 gene showed no mutations in either sibling. Additional intragenic polymorphic marker analysis gave no evidence for different inherited alleles for the 5alpha-reductase 2 gene in the two siblings. Therefore, the absent or reduced expression of 5alpha-reductase 2 is likely to be additional to the AIS. Distinct phenotypic variation in this family was caused by 5alpha-reductase 2 deficiency, additional to AIS. This 5alpha-reductase deficiency is due to absence of expression of the 5alpha-reductase iso-enzyme 2 as shown by molecular studies. The distinct phenotypic variation in AIS here is explained by differences in the availability of 5alpha-dihydrotestosterone during embryonic sex differentiation.

[From gene to disease; androgen receptor gene, androgen insensitivity syndrome, and spinal and bulbar muscle atrophy]. / Van gen naar ziekte; androgeenreceptorgen, androgeenongevoeligheidssyndroom en spinale en bulbaire spieratrofie.

Androgen insensitivity is an X-linked disorder of male sexual differentiation resulting from a defective androgen receptor. Spinal and bulbar muscular atrophy (Kennedy's disease) is an X-linked disease, resulting from expansion of the polyglutamine stretch in the N-terminal part of the androgen receptor. Mutation analysis confirms the clinical diagnosis of androgen insensitivity and enables carrier detection and prenatal diagnosis. Kennedy's disease, with its diagnostic problem of clinical variability, is diagnosed or excluded when an expanded CAG-repeat is present or absent in exon 1 of the androgen receptor. Molecular testing can be used for carrier detection and genetic counselling.

Hormonal control of gubernaculum development during testis descent: gubernaculum outgrowth in vitro requires both insulin-like factor and androgen.

The gubernaculum connects the gonad to the inguinoscrotal region and is involved in testis descent. It rapidly develops in the male fetus, whereas development in the female fetus is lacking. Possible factors involved in gubernaculum development are androgens, anti-Müllerian hormone (AMH), and insulin-like factor (Insl3). Sexual dimorphism in gubernaculum development correlated with the mitotic activity of cells in the gubernacular bulbs from male and female fetuses. Androgen receptor expression was restricted to the mesenchymal core of the gubernacular bulb, whereas skeletal muscle was detected in its outer layer. In an organ culture system devised to further study gubernaculum development in vitro, morphology of gubernacular explants grown in the presence of testes was comparable with that of gubernacula developed in vivo. Testicular tissue or medium containing R1881, a synthetic androgen, had a growth stimulatory effect on gubernacular explants compared with ovarian tissue or basal medium only. Moreover, Amh-/-, Amh+/-, and Insl3+/- testes stimulated the growth of gubernacular explants to the same extent as control testes. Insl3-/- testes, however, did not produce such an activity. This study reveals an essential role for both androgen and Insl3 in the gubernaculum outgrowth during transabdominal testis descent.

Characterization of a zinc-finger protein and its association with apoptosis in prostate cancer cells.

BACKGROUND: The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product. METHODS: The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided. RESULTS: Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells. CONCLUSIONS: We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer.

Gene expression profiles of human endometrial cancer samples using a cDNA-expression array technique: assessment of an analysis method.

The recently developed cDNA expression array technique can be used to generate gene-expression fingerprints of tumour specimens. To gain insight into molecular mechanisms involved in the development and progression of cancer, this cDNA expression array technique could be a useful tool, however, no established methods for interpreting the results are yet available. We used the Atlas cancer cDNA expression array (Clontech, USA) for analysing total RNA isolated from four human endometrial carcinoma samples (two cell-lines and two tissue samples), one benign endometrial tissue sample and a human breast cancer cell-line, in order to develop a method for analysing the array data. The obtained gene-expression profiles were highly reproducible. XY-scatterplots and regression analysis of the logarithmic transformed data provided a practical method to analyse the data without the need of preceding normalization. Three genes (Decorin, TIMP3 and Cyclin D1) were identified to be differentially expressed between the benign endometrial tissue sample and the endometrial carcinoma samples (tissue and cell-lines). These three genes may potentially be involved in cancer progression. A higher degree of similarity in gene-expression profile was found between the endometrial samples (tissue and cell-lines) than between the endometrial samples and the breast cancer cell-line, which is indicative for an endometrial tissue-specific gene-expression profile.