Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding.

This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is predominantly confined to a single cell population, i.e. the choanocytes, and in this process apoptosis only plays a minor role. To our knowledge, such fast cell kinetics under steady-state conditions, with high turnover by shedding in the absence of apoptosis, has not been observed previously in any other multicellular organism. The duration of the cell cycle in vivo resembles that of unicellular organisms in culture. Morphological and histochemical studies demonstrate compartmentalization of choanocytes in the sponge tissue, which corresponds well with its remarkable cellular kinetics. Coral reef cavity sponges, like H. caerulea, inhabit low nutrient tropical waters, forcing these organisms to filter large volumes of water and to capture the few nutrients efficiently. Under these oligotrophic conditions, a high cell turnover may be considered as a very useful strategy, preventing permanent damage to the sponge by environmental stress. Halisarca caerulea maintains its body mass and keeps its food uptake system up to date by constantly renewing its filter system. We conclude that studies on cell kinetics and functional morphology provide new and essential information on the growth characteristics and the regulation of sponge growth in vivo as well as in vitro and the role of choanocytes in tissue homeostasis.

MGMT and MLH1 promoter methylation versus APC, KRAS and BRAF gene mutations in colorectal cancer: indications for distinct pathways and sequence of events.

BACKGROUND: To study how caretaker gene silencing relates to gatekeeper mutations in colorectal cancer (CRC), we investigated whether O6-methylguanine DNA methyltransferase (MGMT) and Human Mut-L Homologue 1 (MLH1) promoter hypermethylation are associated with APC, KRAS and BRAF mutations among 734 CRC patients. METHODS: We compared MGMT hypermethylation with G:C > A:T mutations in APC and KRAS and with the occurrence of such mutations in CpG or non-CpG dinucleotides in APC. We also compared MLH1 hypermethylation with truncating APC mutations and activating KRAS and BRAF mutations. RESULTS: Only 10% of the tumors showed both MGMT and MLH1 hypermethylation. MGMT hypermethylation occurred more frequently in tumors with G:C > A:T KRAS mutations (55%) compared with those without these mutations (38%, P < 0.001). No such difference was observed for G:C > A:T mutations in APC, regardless of whether mutations occurred in CpG or non-CpG dinucleotides. MLH1 hypermethylation was less common in tumors with APC mutations (P = 0.006) or KRAS mutations (P = 0.001), but was positively associated with BRAF mutations (P < 0.001). CONCLUSIONS: MGMT hypermethylation is associated with G:C > A:T mutations in KRAS, but not in APC, suggesting that MGMT hypermethylation may succeed APC mutations but precedes KRAS mutations in colorectal carcinogenesis. MLH1-hypermethylated tumors harbor fewer APC and KRAS mutations and more BRAF mutations, suggesting that they develop distinctly from an MGMT methylator pathway.

Effects of selective oestrogen receptor modulators on proliferation in tissue cultures of pre- and postmenopausal human endometrium.

We characterised the effects of selective oestrogen receptor modulators (SERM) in explant cultures of human endometrium tissue. Endometrium tissues were cultured for 24h in Millicell-CM culture inserts in serum-free medium in the presence of vehicle, 17beta-estradiol (17beta-E2, 1nM), oestrogen receptor (ER) antagonist ICI 164.384 (40nM), and 4-OH-tamoxifen (40nM), raloxifene (4nM), lasofoxifene (4nM) and acolbifene (4nM). Protein expression of ERalpha, ERbeta1 and Ki-67 were evaluated by immunohistochemistry (IHC). The proliferative fraction was assessed by counting the number of Ki-67 positive cells. Nuclear staining of ER( and ER(1 was observed in the glandular epithelium and stroma of pre- and postmenopausal endometrium. ER(1 protein was also localized in the endothelial cells of blood vessels. Treating premenopausal endometrium tissue with 17beta-E2 increased the fraction of Ki-67 positive cells (p<0.001) by 55% in glands compared to the control. Raloxifene (4nM) increased (p<0.05) the Ki-67 positive fraction. All other SERMS did not affect proliferation in this model. Treating postmenopausal endometrium with 17(-E2 increased (p<0.001) the fraction of Ki-67 positive cells by 250% in glands compared to the control. A similar effect was also seen for 4-OH-tamoxifen, whereas the rest of SERMs did not stimulate proliferation. We demonstrated that oestradiol increases the fraction of proliferating cells in short term explant cultures of postmenopausal endometrium. In addition, we were able to reveal the agonistic properties of 4-OH-tamoxifen and confirm that raloxifene and next-generation SERMs acolbifene and lasofoxifene were neutral on the human postmenopausal endometrium.

Cigarette smoking and KRAS oncogene mutations in sporadic colorectal cancer: results from the Netherlands Cohort Study.

Since a KRAS oncogene mutation is an early event in colorectal cancer development and cigarette smoking is thought to have an effect on early stages of colorectal tumorigenesis, smoking, especially long-term smoking, may be associated with the risk for colorectal cancer with KRAS oncogene mutations. In the Netherlands Cohort Study on diet and cancer (n=120,852 men and women), using a case-cohort design, adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) were computed for colorectal tumors with wild-type and with mutated KRAS gene, and with specific G:C-->T:A or G:C-->A:T point mutations in KRAS, according to cigarette smoking status, frequency, duration, pack years, age at first exposure, years since cessation, inhalation and filter usage. After 7.3 years and excluding the first 2.3 years, 648 cases and 4083 sub-cohort members were included in the analyses. Ex-smokers, but not current smokers, were at increased risk for colorectal cancer with wild-type KRAS gene tumors when compared with never smokers, albeit not statistically significant (RR 1.26, 95% CI 0.96-1.66). This was not observed for KRAS mutated tumors when comparing ex-smokers with never smokers (RR 1.15, 95% CI 0.79-1.66). The highest category of smoking frequency (>20 cigarettes/day) and inhalation of smoke were associated with an increased risk for colorectal cancer with wild-type KRAS gene tumors, though not statistically significant, when compared with never smoking (frequency: RR 1.24, 95% CI 0.90-1.71 and inhalation: RR 1.25, 95% CI 0.94-1.67). These associations were strongest in men (ex-smokers: RR 1.79, 95% CI 1.00-3.20; frequency: RR 1.91, 95% CI 1.03-3.52; inhalation: RR 1.69, 95% CI 0.94-3.04). No associations were observed between any of the smoking characteristics and the risk for colorectal cancer with mutated KRAS gene tumors, nor where there any clear associations with tumors with specific G:C-->A:T transitions or G:C-->T:A transversions. These results suggest that, in contrast to the hypothesis, smoking does not increase the risk for colorectal tumors with a mutated KRAS gene. Some smoking characteristics, i.e. being an ex-smoker, frequency and inhalation, may be associated with risk for colorectal cancer characterized by the wild-type KRAS gene, especially in men.

Haemoglobin expression in human endometrium.

BACKGROUND: The general concept that haemoglobin is only a carrier protein for oxygen and carbon dioxide is challenged since recent studies have shown haemoglobin expression in non-erythroid cells and the protection of haemoglobin against oxidative and nitrosative stress. Using microarrays, we previously showed expression of haemoglobins alpha, beta, delta and gamma and the haeme metabolizing enzyme, haeme oxygenase (HO)-1 in human endometrium. METHODS: Using real-time quantitative PCR, haemoglobin alpha, beta, delta and gamma, and HO-1 mRNA levels were assessed throughout the menstrual cycle (n = 30 women). Haemoglobin and HO-1 protein levels in the human endometrium were assessed with immunohistochemistry. For steroid responsiveness, menstrual and late proliferative-phase endometrial explants were cultured for 24 h in the presence of vehicle (0.1% ethanol), estradiol (17beta-E(2,) 1 nM), progestin (Org 2058, 1 nM) or 17beta-E(2)+Org 2058 (1 nM each). RESULTS: All haemoglobins and the HO-1 were expressed in normal human endometrium. Haemoglobin mRNA and protein expression did not vary significantly during the menstrual cycle. Explant culture with Org 2058 or 17beta-E(2)+Org 2058 increased haemoglobin gamma mRNA expression (P < 0.05). HO-1 mRNA levels, and not protein levels, were significantly higher during the menstrual (M)-phase of the cycle (P < 0.05), and were down-regulated by Org 2058 in M-phase explants and by 17beta-E(2)+Org 2058 in LP-phase explants, versus control (P < 0.05). CONCLUSIONS: The haemoglobin-HO-1 system may be required to ensure adequate regulation of the bioavailability of haeme, iron and oxygen in human endometrium.

Progesterone regulation of implantation-related genes: new insights into the role of oestrogen.

Genomic profiling was performed on explants of late proliferative phase human endometrium after 24-h treatment with progesterone (P) or oestradiol and progesterone (17beta-E(2)+P) and on explants of menstrual phase endometrium treated with 17beta-E(2)+P. Gene expression was validated with real-time PCR in the samples used for the arrays, in endometrium collected from early and mid-secretory phase endometrium, and in additional experiments performed on new samples collected in the menstrual and late proliferative phase. The results show that late proliferative phase human endometrium is more responsive to progestins than menstrual phase endometrium, that the expression of several genes associated with embryo implantation (i.e. thrombomodulin, monoamine oxidase A, SPARC-like 1) can be induced by P in vitro, and that genes that are fully dependent on the continuous presence of 17beta-E(2) during P exposure can be distinguished from those that are P-dependent to a lesser extent. Therefore, 17beta-E(2) selectively primes implantation-related genes for the effects of P.

Tibolone and metabolites induce prolactin production in human endometrial stromal cells in vitro: evidence for cell-specific metabolism.

In this study, we assessed the effects of tibolone and its metabolites on the production of a progesterone sensitive parameter, prolactin, in human endometrium stroma cells in vitro. In addition, the metabolism of the compounds by isolated stromal and epithelial cells was evaluated. The reference compounds, progesterone, Org 2058, and DHT all induced prolactin production. Oestradiol also slightly induced prolactin production and enhanced the response to Org 2058. Tibolone and Delta4-tibolone were similar with regard to potency to induce prolactin levels in the culture supernatant. Their potency was lower than that of Org 2058, similar to that of progesterone and higher than that of DHT. The efficacies of tibolone, Delta4-tibolone and Org 2058 were similar (approximately 200-fold induction). The estrogenic tibolone metabolites 3alpha- and 3beta-OH tibolone also significantly stimulated prolactin production. Their potency, however, was low since significance was reached only at the highest concentrations tested. The PR antagonist Org 31710 inhibited both tibolone- and Delta4-tibolone-induced prolactin production. The responses of tibolone and Delta4-tibolone were not affected by co-incubation with the androgen receptor antagonist OH-flutamide. The effect of tibolone, but not Delta4-tibolone, was antagonized approximately 50% in combination with the highest dose (1 microM) estrogen receptor antagonist, ICI 164384. The induction of prolactin by 3alpha- and 3beta-OH tibolone was antagonized most potently by Org 31710, but also by ICI 164384 and OH-flutamide. Tibolone is metabolized differently in epithelial and stromal cells of the human endometrium. The epithelial cells mostly produce the progestagenic/androgenic Delta4-tibolone. The stromal cells produce predominantly the 3beta-OH tibolone, and some Delta4-tibolone, but the net effect observed with regard to prolactin production is progestagenic. When the metabolites 3alpha-OH, 3beta-OH, and Delta4-tibolone were added to the cultures no conversions were observed. The HPLC analyses showed no evidence for the production of sulfated metabolites. In conclusion, the net effects on endometrial stromal cells are predominantly progestagenic. Tibolone is converted by epithelial cells into Delta4-tibolone which displays progestagenic and androgenic activities, whereas in stromal cells also the estrogenic metabolites 3alpha- and 3beta-OH tibolone are formed.

Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium.

Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6-8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1alpha and CA-IX in the menstrual and early proliferative phases. HIF1alpha was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed by estrogen during the late proliferative phase.

Meat consumption and K-ras mutations in sporadic colon and rectal cancer in The Netherlands Cohort Study.

Case-cohort analyses were performed on meat and fish consumption in relation to K-ras mutations in 448 colon and 160 rectal cancers that occurred during 7.3 years of follow-up, excluding the first 2.3 years, and 2948 subcohort members of The Netherlands Cohort Study on diet and cancer. Adjusted incidence rate ratios and 95% confidence intervals were computed for colon and rectal cancer and for K-ras mutation status subgroups. Total fresh meat, most types of fresh meat and fish were not associated with colon or rectal cancer, neither overall nor with K-ras mutation status. However, several weak associations were observed for tumours with a wild-type K-ras, including beef and colon tumours, and an inverse association for pork with colon and rectal tumours; for meat products, an increased association was observed with wild-type K-ras tumours in the colon and possibly with G>A transitions in rectal tumours.

Oestrogen-modulated gene expression in the human endometrium.

To identify key regulatory mechanisms in the growth and development of the human endometrium, microarray analysis was performed on uncultured human endometrium collected during menstruation (M) and the late-proliferative (LATE-P)-phase of the menstrual cycle, as well as after 24 h incubation in the presence of oestradiol (17beta-E2). We demonstrate the expression of novel gene transcripts in the human endometrium. i.e. mucin-9, novel oestrogen-responsive gene transcripts, i.e. gelsolin and flotillin-1, and genes known to be expressed in human endometrium but not yet shown to be oestrogen responsive, i.e. connexin-37 and TFF1/pS2. Genes reported to be expressed during the implantation window and implicated in progesterone action, i.e. secretoglobin family 2A, member 2 (mammaglobin) and homeobox-containing proteins, were up-regulated in uncultured LATE-P-phase endometrium compared to M-phase endometrium. Some gene transcripts are regulated directly by 17beta-E2 alone, others are influenced by the in vivo environment as well. These observations emphasise that the regulation of endometrium maturation by oestrogen entails more then just stimulation of cell proliferation.

APC, beta-catenin, and E-cadherin and the development of recurrent endometrial carcinoma.

Endometrial carcinoma, generally, has a good prognosis. However, in some patients, the tumor appears to behave very aggressively, a course that cannot be explained with histopathological characteristics. More insight into the molecular background can be valuable to clarify these differences in tumor behavior. The three components associated with the Wnt pathway--i.e., adenomatous polyposis coli (APC), beta-catenin, and E-cadherin--were evaluated in a case-control study of 28 patients with stage-I endometrial carcinomas to determine their involvement in the development of recurrent disease. Mutation analysis of the mutation cluster region of the APC gene, determination of gene promoter methylation status of the APC-1A and E-cadherin genes, and immunohistochemical analysis of APC, E-cadherin, and beta-catenin were performed using paraffin-embedded tumor tissue. Twenty-one APC gene mutations were detected in 12 of 28 (43%) patients. Only three mutations would result in a stopcodon in the APC gene. APC gene promoter methylation was assessed in 12 of 28 (43%) patients. APC immunostaining was absent in two of 24 (8.3%) patients. The occurrence of APC mutations, APC gene promoter methylation, and APC immunostaining were not predictive for recurrence. No E-cadherin expression was observed in four of 24 patients (17%). E-cadherin gene promoter methylation could not be detected in any of the patients. The absence of E-cadherin expression was predictive for distant metastases, but not for local recurrence. Nuclear localization of beta-catenin was present in nine of 24 (38%) patients and was not predictive for recurrent disease. Involvement of epigenetic and genetic aberrations in APC and beta-catenin genes seems to be of minor importance for the development of local recurrences and distant metastases. Although the number of patients is limited, E-cadherin expression appears to be predictive for the development of distant metastases in endometrial carcinoma.

Menstrual effluent induces epithelial-mesenchymal transitions in mesothelial cells.

BACKGROUND: Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). METHODS: Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. RESULTS: Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and alpha-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. CONCLUSIONS: In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Endometrial angiogenesis throughout the human menstrual cycle.

BACKGROUND: The timing and mechanisms of new blood vessel formation in the endometrium during the menstrual cycle are still largely unknown. In the present study we used the chick embryo chorioallantoic membrane (CAM) as an in-vivo assay for angiogenesis to assess the angiogenic potential of endometrium obtained at different stages of the menstrual cycle. METHODS: Endometrial fragments were explanted onto the CAM and, after 4 days of incubation, slides of the treated area were taken in ovo through a microscope for computerized image analysis. The vascular density index (VDI), a stereological estimate of vessel number and length, was obtained by counting the intersections of vessels with five concentric circles of a circular grid superimposed on the computerized image. RESULTS: We demonstrated that human endometrium has angiogenic potential throughout the menstrual cycle. Furthermore, there was a significant difference in angiogenic response between the stages of the menstrual cycle (P = 0.01). The VDIs of the early proliferative, early and late secretory stage were significantly higher than the VDI of the late proliferative phase. CONCLUSIONS: Elongation of existing vessels during the early proliferative phase as well as growth and coiling of the spiral vessels during the secretory phase may demand far higher angiogenic activity than outgrowth and maintenance of vessels during the late proliferative phase.

The Mesothelium, Teflon or Velcro? Mesothelium in endometriosis pathogenesis.

Sampson's transplantation theory for the pathogenesis of peritoneal endometriosis is widely accepted. The events that take place, however, on the cellular and subcellular level during the transition of endometrial tissue in the abdominal cavity into peritoneal endometriosis remain controversial. The mesothelium plays a central role in the debate on this subject. The interaction between endometrium and peritoneum has been studied in an in-vitro model using amnion, peritoneum and mesothelial cells in culture on the one hand and cyclic and menstrual endometrium on the other hand. The results of these studies indicate that (i) an intact mesothelial lining prevents adhesion of shed endometrial tissue, (ii) shed endometrial tissue adheres to the peritoneal extracellular matrix and (iii) menstrual effluent creates its own adhesion sites by damaging the mesothelial lining thus exposing the extracellular matrix. Therefore we conclude that the mesothelium has the properties of Teflon, while the extracellular matrix resembles Velcro.

Development of endometriosis-like lesions after transplantation of human endometrial fragments onto the chick embryo chorioallantoic membrane.

The chick embryo chorioallantoic membrane (CAM) bioassay was used to investigate the early pathogenesis of endometriosis. Endometrial fragments were explanted onto the CAM. The grafts including the surrounding CAM were excised at 24, 48 or 72 h after explantation, fixed and embedded in paraffin. Immunohistochemical analysis was used to distinguish endometrial cells. To identify cells of human origin, in-situ hybridization was performed using a probe specific for human chromosome 1. After 24 h, direct contact between endometrial stromal as well as epithelial cells and the mesenchymal layer of the CAM was observed. Invasion of both stromal cells and intact endometrial glands into the mesenchymal layer was observed after 48 h. At 72 h, endometriosis-like lesions were observed in the mesenchymal layer. Positive staining with antibodies to vimentin and pan-cytokeratin was observed in the invading cells as well as in the lesions. In the lesions these positively stained cells showed in-situ hybridization signals for human chromosome 1, confirming their human origin. In conclusion, after 3 days of incubation, endometriosis-like lesions consisting of human endometrial glands and stromal cells were found in the mesenchymal layer of the CAM. These lesions apparently resulted from the invasion of intact human epithelial structures and stromal cells.

Molecular genetic alterations in hamartomatous polyps and carcinomas of patients with Peutz-Jeghers syndrome.

AIM: To investigate whether mutations in the STK11/LKB1 gene and genes implicated in the colorectal adenoma-carcinoma sequence are involved in Peutz-Jeghers syndrome (PJS) related tumorigenesis. METHODS: Thirty nine polyps and five carcinomas from 17 patients (from 13 families) with PJS were analysed for loss of heterozygosity (LOH) at 19p13.3 (STK11/LKB1 gene locus), 5q21 (APC gene locus), 18q21-22 (Smad4 and Smad2 gene locus), and 17p13 (p53 gene locus), and evaluated for immunohistochemical staining of p53. In addition, mutational analysis of K-ras codon 12, APC, and p53 and immunohistochemistry for Smad4 expression were performed on all carcinomas. RESULTS: LOH at 19p was seen in 15 of the 39 polyps and in all carcinomas (n = 5). Interestingly, six of the seven polyps from patients with cancer had LOH, compared with nine of the 31 polyps from the remaining patients (p = 0.01). In one polyp from a patient without a germline STK11/LKB1 mutation, no LOH at 19p or at three alternative PJS candidate loci (19q, 6p, and 6q) was found. No LOH at 5q was observed. However, mutational analysis revealed an APC mutation in four of the five carcinomas. LOH at 17p was not seen in polyps or carcinomas; immunohistochemistry showed expression of p53 in one carcinoma and focal expression in three polyps. At subsequent sequence analysis, no p53 mutation was found. One carcinoma had an activating K-ras codon 12 mutation and another carcinoma showed 18q LOH; however, no loss of Smad4 expression was seen. CONCLUSIONS: These results provide further evidence that STK11/LKB1 acts as a tumour suppressor gene, and may be involved in the early stages of PJS tumorigenesis. Further research is needed to see whether LOH in PJS polyps could be used as a biomarker to predict cancer. Differences in molecular genetic alterations noted between the adenoma-carcinoma sequence and PJS related tumours suggest the presence of a distinct pathway of carcinogenesis.

Tumor necrosis factor-alpha but not interleukin-1 beta or interleukin-8 concentrations correlate with angiogenic activity of peritoneal fluid from patients with minimal to mild endometriosis.

OBJECTIVE: To assess the angiogenic activity of peritoneal fluid in women with minimal to mild endometriosis and to investigate the relationship between this activity and the concentration of macrophage-derived angiogenic factors and clinical variables, such as phase of menstrual cycle, type of lesion, and revised American Society for Reproductive Medicine classification. DESIGN: In vivo bioassay. SETTING: Tertiary-care university medical center. PATIENT(S): Fifty-two female volunteers with laparoscopic findings indicating minimal to mild endometriosis. INTERVENTION(S): Peritoneal fluid was collected at the start of laparoscopy. A standard amount of peritoneal fluid was applied to a chick embryo chorioallantoic membrane assay. MAIN OUTCOME MEASURE(S): Angiogenic response was assessed by determining the vascular density index. RESULT(S): 85% of the peritoneal fluid samples induced angiogenesis in the chick embryo chorioallantoic membrane bioassay. Tumor necrosis factor-alpha and total protein were significantly related to the vascular density index, whereas interleukin-1beta, interleukin-8, and clinical variables appeared to not affect the angiogenic response. CONCLUSION(S): The results confirms previous findings of peritoneal fluid angiogenic activity in women with minimal to mild endometriosis and indicate involvement of tumor necrosis factor-alpha.