Distinct roles for S100a8 in early embryo development and in the maternal deciduum.

S100a8 is a cytosolic protein expressed in myeloid cells where it forms a stable heterodimer with another S100 protein family member, S100a9. The S100a9(-/-) mouse is viable and phenotypically normal, whereas the S100a8(-/-) condition is embryonic lethal. We present evidence that S100a8, without S100a9, has a previously unrecognized role in embryo development between fertilization and the 8-cell stage at embryonic day (E) 2.5. S100a8 also has a second role in the maternal deciduum, where expression is associated with the vasculature from the E8.5 stage to the formation of mature placenta. Uterine natural killer cells that have a role in vascular remodelling colocalise with the S100a8 vascular expression in the metrial triangle. In inflammatory responses in peripheral tissues, S100a8 is a potent chemoattractant and also an anti-oxidant. Both roles may be important in the developing placenta. Thus we highlight two new S100a9-independent roles for S100a8 in early embryo development.

CUX1: target of Akt signalling and mediator of resistance to apoptosis in pancreatic cancer.

BACKGROUND AND AIMS: The transcription factor CUX1 is known as a regulator of cell differentiation and cell cycle progression. Previously, CUX1 was identified as a modulator of invasiveness in various cancers. Based on expression profiles suggesting a role for CUX1 in mediating chemoresistance, the aim of this study was to characterise the effect of CUX1 on apoptosis as well as its regulation by signalling pathways modulating drug resistance in pancreatic cancer. METHODS: The effect of CUX1 on TRAIL- (tumour necrosis factor-related apoptosis-inducing ligand) and drug-induced apoptosis was analysed using overexpression and knock-down strategies. Regulation of CUX1 by phosphatidylinositol-3-kinase (PI3K)/Akt signalling was examined at the mRNA and protein level. The effect of CUX1 knock-down by nanoparticle-complexed small interfering RNA (siRNA) in vivo was analysed in a murine xenograft model. Furthermore, CUX1 RNA and protein expression was evaluated in human pancreatic cancer and adjacent normal tissues. RESULTS: Knock-down of CUX1 resulted in significantly enhanced TRAIL- and drug-induced apoptosis, associated with increased PARP (poly ADP-ribose polymerase) cleavage and caspase activity. Vice versa, overexpression of CUX1 inhibited apoptosis. CUX1 expression was induced by activation of Akt/protein kinase B signalling, and decreased by PI3K inhibitors. The antiapoptotic effect of CUX1 was associated with upregulation of BCL2 and downregulation of tumour necrosis factor alpha. CUX1 was significantly overexpressed in pancreatic cancers, as analysed by in situ hybridisation and immunohistochemistry. In vivo, silencing of CUX1 by intratumourally administered polyethylenimine-complexed siRNA led to reduced tumour growth and increased apoptosis in pancreatic cancer xenografts. CONCLUSION: CUX1 was identified as an important mediator of tumour cell survival in pancreatic cancer in vitro and in vivo.

mRNA expression profiling of phyllodes tumours of the breast: identification of genes important in the development of borderline and malignant phyllodes tumours.

The aim of this study was to identify genes involved in the development of borderline and malignant phyllodes tumours of the breast (PTs). Expression profiling of 23 PTs (12 benign, 11 borderline/malignant) was performed using Affymetrix U133A GeneChips. mRNA expression in the borderline/malignant PTs was compared to the benign PTs. A group of 162 genes was over-expressed in the borderline/malignant group with a fold change > 2 and FDR < 0.1. Four of these genes were chosen for further investigation: PAX3, SIX1, TGFB2 and HMGA2. Over-expression was validated in a separate set of formalin-fixed, paraffin-embedded (FFPE) tumours, using either in situ hybridization or immunohistochemistry. This confirmed that expression of PAX3, SIX1, TGFB2 and HMGA2 in the stromal component of PTs was associated with the borderline/malignant phenotypes (p = 8.7 x 10(-5), p = 0.05, p = 0.009, p = 0.003, respectively; Fisher's exact test). The functional consequences of down-regulating these genes were studied using siRNA in short-term cultures and cell lines established from PTs. mRNA 'knock-down' of PAX3 resulted in significantly decreased cell proliferation in both a malignant and a borderline PT cell culture. mRNA 'knock-down' of SIX1 and HMGA2 resulted in decreased cell proliferation only in the malignant PT cell line, and 'knock-down' of TGFB2 resulted in decreased cell proliferation only in the borderline PT cell culture. This study shows that these four genes are involved in the development of borderline/malignant PTs. SIX1 over-expression was most marked in the highly malignant PTs, with particularly high expression in one case of metastatic PT. PAX3, TGFB2 and HMGA2 were expressed predominantly in borderline/malignant PTs, but showed some expression in benign tumours; they may be important in the transition from the benign to borderline/malignant phenotype.

The cellular origin and proliferative status of regenerating renal parenchyma after mercuric chloride damage and erythropoietin treatment.

OBJECTIVES: In this study, we have sought to establish the cellular origin and proliferative status of the renal parenchyma as it regenerates after damage induced by mercuric chloride, with or without erythropoietin treatments, that might alter the response. MATERIALS AND METHODS: Female mice were irradiated and male whole bone marrow was transplanted into them. Six weeks later recipient mice were assigned to one of four groups: control, mercuric chloride treated, erythropoietin treated and treated with mercuric chloride plus erythropoietin. RESULTS: Tubular injury scores were high 3 days after mercuric chloride and had recovered partially after 14 days, in line with serum urea nitrogen levels. Confocal microscopy confirmed the tubular location of bone marrow-derived cells. A 'four-in-one' analytical technique (identifying cell origin, tubular phenotype, tubular basement membranes and S-phase status) revealed that tubular necrosis increased bone marrow derivation of renal tubular epithelium from a baseline of approximately 1.3% to approximately 4.0%. Erythropoietin increased the haematocrit, but no other effects were detected. CONCLUSION: As 1 in 12 proximal tubular cells in S-phase was derived from bone marrow, we conclude that in the kidney, the presence of bone marrow-derived cells makes a minor but important regenerative contribution after tubular necrosis.

Interactions between stromal cell--derived keratinocyte growth factor and epithelial transforming growth factor in immune-mediated crypt cell hyperplasia.

Immune reactions in the gut are associated with increased epithelial cell proliferation. Here we have studied the role of keratinocyte growth factor (KGF; FGF7) and transforming growth factor-alpha (TGF-alpha) in the epithelial cell hyperplasia seen in explants of fetal human small intestine after activation of lamina propria T cells with the superantigen Staphylococcus aureus enterotoxin B (SEB). After the addition of SEB to the explants there is a 10-fold increase in KGF mRNA by 72 h of culture. KGF transcripts were abundant in the lamina propria using in situ hybridization and the culture supernatants contained elevated amounts of KGF protein. SEB had no direct effect on KGF mRNA and protein production by cultured lamina propria mesenchymal cells, but both were upregulated by TNF-alpha. Accompanying the increase in KGF there was also an increase in TGF-alpha precursor proteins in the culture supernatants and the phosphorylated form of the EGFR receptor was also detected in the tissue. Increased TGF-alpha precursor proteins were also detected in the supernatants of control explants stimulated with KGF alone. The direct addition of KGF and TGF-alpha enhanced epithelial cell proliferation and antibodies against KGF and TGF-alpha partially inhibited SEB-induced crypt hyperplasia. These results suggest molecular cross-talk between the KGF/KGFR and the TGF-alpha/EGFR in immune-mediated crypt cell hyperplasia.

Expression of the Met/hepatocyte growth factor receptor in human pancreatic cancer.

The c-MET oncogene encodes the receptor for hepatocyte growth factor (HGF) scatter factor, a multifunctional cytokine able to mediate morphogenesis as well as invasive growth of epithelial cells. The c-MET-encoded receptor is detectable only at low levels in the normal human exocrine pancreas, but it is up-regulated in the majority of pancreatic ductal adenocarcinomas. The c-MET-encoded HGF receptor is also overexpressed in a proportion of the panel of 31 human pancreatic cancer cell lines examined, which have a range of different growth properties and degrees of differentiation. In most cases the HGF receptor found in the malignant cells has features of the normal receptor. When added to pancreatic cancer cell lines, HGF triggers receptor phosphorylation and stimulates cells to move and proliferate. In overexpressing cell lines, the Met/HGF receptor is phosphorylated in the absence of endogenously produced or exogenously added ligand. These data suggest that the Met/HGF receptor may be involved in the growth and behavior of pancreatic cancer and may contribute to the ductal phenotype of these tumors.

Allelic loss at SMAD4 in polyps from juvenile polyposis patients and use of fluorescence in situ hybridization to demonstrate clonal origin of the epithelium.

Juvenile polyposis syndrome (JPS; Online Mendelian Inheritance in Man2 174900) is a rare Mendelian disorder in which individuals have typical hamartomatous polyps within the gastrointestinal tract. The stromal element of the polyps has classically been thought to be the proliferative component, although epithelial malignancies (largely gastrointestinal cancers) occur more frequently than expected in JPS patients. Germ-line mutations in SMAD4 (DPC4) account for about a third of JPS cases. It has been postulated that the apparent paradox of a stromal lesion predisposing to epithelial malignancy can be resolved by the "landscaper" effect: an abnormal stromal environment affects the development of adjacent epithelial cells, and the resulting regeneration of damaged epithelium leads to an increased risk of cancer. We have found allele loss at the SMAD4 locus on 18q in polyps from JPS individuals with a germ-line SMAD4 mutation, showing that SMAD4 is acting as a tumor suppressor gene in JPS polyps, as it does in sporadic cancers of the gastrointestinal tract. Interphase fluorescence in situ hybridization showed deletion of one copy of SMAD4 in the epithelial component of JPS polyps, but not in the inflammatory infiltrate. Fluorescence in situ hybridization also suggested that a single copy of SMAD4 was present in stromal fibroblasts of JPS polyps. Thus, biallelic inactivation of SMAD4 occurs in both the epithelium and some of the stromal cells in these lesions, suggesting a common clonal origin. Epithelial malignancies almost certainly develop in juvenile polyposis through direct malignant progression of the epithelial component of the hamartomas. SMAD4/DPC4 probably acts as a "gatekeeper" tumor suppressor in juvenile polyps, and there is no need to invoke a "landscaper hypothesis."

The cloning, mapping and expression of a novel gene, BRL, related to the AF10 leukaemia gene.

The MLL gene is reciprocally translocated with one of a number of different partner genes in a proportion of human acute leukaemias. The precise mechanism of oncogenic transformation is unclear since most of the partner genes encode unrelated proteins. However, two partner genes, AF10 and AF17 are related through the presence of a cysteine rich region and a leucine zipper. The identification of other proteins with these structures will aid our understanding of their role in normal and leukaemic cells. We report the cloning of a novel human gene (BRL) which encodes a protein containing a cysteine rich region related to that of AF10 and AF17 and is overall most closely related to the previously known protein BR140. BRL maps to chromosome 22q13 and shows high levels of expression in testis and several cell lines. The deduced protein sequence also contains a bromodomain, four potential LXXLL motifs and four predicted nuclear localization signals. A monoclonal antibody raised to a BRL peptide sequence confirmed its widespread expression as a 120 Kd protein and demonstrated localization to the nucleus within spermatocytes.

Analysis of foetal expression sites of human type II DNA topoisomerase alpha and beta mRNAs by in situ hybridisation.

We present the first analysis of the sites of expression of DNA topoisomerase II alpha and II beta mRNAs in human foetal tissues by in situ hybridisation, using 35S-radiolabelled probes. This revealed differential localisation of topoisomerase II alpha and II beta mRNAs in a range of foetal organs, including foetal kidney (developing structures within the neogenic zone), brain (cortical layers), small intestine (crypt epithelium and muscle), liver (hepatocytes), lung (smooth muscle, and epithelium in the lining of primitive lung buds) and placenta (trophoblastic epithelium). The intensity of expression of topoisomerase II alpha mRNA appeared higher than that of topoisomerase II beta, although topoisomerase II beta mRNA was expressed in a broader range of cell types. The distinct patterns of expression of topoisomerase II alpha and beta mRNAs indicate differential regulation of these genes, suggesting that the two isoforms may play important but different roles in foetal development, with topoisomerase II alpha being expressed most strongly in zones of proliferation.

Repopulation of human pulmonary epithelium by bone marrow cells: a potential means to promote repair.

After lung injury and damage to the alveolar epithelium, the underlying basement membranes become exposed. Proliferation of type II pneumocytes and their differentiation to the type I phenotype have been considered to be the mechanism by which repopulation of the alveolar epithelium occurs. A growing body of evidence has shown that tissues can be repaired by cells acquired via the circulation. For the lung, bone marrow stem cells have been shown in mice to regenerate epithelium as well as give rise to the expected mesodermal derivatives. We hypothesized that extrapulmonary cells, including those from the bone marrow, can contribute to the reepithelialization of human alveoli. To investigate this, we examined samples of peripheral lung from patients who had undergone cross-gender transplantation of lung or bone marrow. Thus, archival blocks of peripheral lung were analyzed from male patients (surgical samples, n = 8) who had received a lung transplant from a female donor and female patients (postmortem samples, n = 3) who had male bone marrow transplants. In both cases, male cells were identified in the female lungs by Y chromosome in situ hybridization. Male cells could be identified in the alveolar epithelium where, in the better preserved, transplanted lungs, it was possible to show that some had differentiated to type II pneumocytes. In addition, Y chromosomes were found to be widespread in cells of mesenchymal lineage, including macrophages and endothelial cells. Concomitant visualization of Y and X chromosomes, using fluorescence immunolabeling, yielded no evidence of cellular fusion, although the poor quality of the autopsy samples studied meant that the possibility could not be excluded. These observations suggest that, as occurs in rodents, the epithelium of the adult human lung has the capacity to renew itself, using cells recruited from extrapulmonary sources, including the bone marrow. This finding could provide new therapeutic opportunities for a range of pulmonary diseases by providing means to repair the lung and a novel route for gene therapy.

Wnt5a cloning, expression, and up-regulation in human primary breast cancers.

Wnt genes are involved in mouse mammary cancer, but their role in human cancer is unknown. Human Wnt5a was cloned from a placental cDNA library and used to assess expression by ribonuclease protection and in situ hybridization in human breast cell lines and in normal, benign, and malignant breast tissues. Human Wnt5a shows over 99% homology at amino acid level with mouse Wnt5a, and 90% with Xenopus Wnt5a. It was expressed only at low levels in breast cell lines and normal breast tissue. Benign proliferations and invasive cancer respectively showed 10-fold and 4-fold higher Wnt5a than normal breast tissues. The greater up-regulation in benign conditions suggests a role in aberrant differentiation. In situ hybridization localized the signal to the epithelial component. Wnt5a is the first member of the Wnt family to demonstrate overexpression in human breast cancer. It was not associated with factors known to affect breast cancer prognosis such as lymph node status or epidermal growth factor receptor status.

The gene encoding mouse intestinal trefoil factor: structural organization, partial sequence analysis and mapping to murine chromosome 17q.

Trefoil peptides, a growing family of secretory molecules, have been identified mainly in the gastrointestinal tract of humans, rodents and amphibians. In the present study, the nucleotide sequence of a large portion (81%) of the gene encoding murine intestinal trefoil factor (mITF) and its whole genomic organization were determined. The mITF gene contains three exons distributed over 5 kb of genomic DNA. The genomic sequence is highly conserved, as compared with that of the rat and human ITF, and contains several AP-1-binding sites, the consensus binding site for the transcription factor Sp1, and a sequence homologous to a heat-shock element. Fluorescence in situ hybridization was used to assign ITF to chromosome 17 of the murine genome, a region syntenic with the trefoil gene cluster on human chromosome 21q22.3.

Primary mucinous carcinomas of the skin express TFF1, TFF3, estrogen receptor, and progesterone receptors.

Mucinous carcinoma may present at various sites, including the breast and the gastrointestinal tract. Rarely, such tumors arise within the skin. Comparatively, breast lesions are relatively common and usually associated with a good prognosis. When pure, they are typically estrogen (ER) and progesterone receptor (PR) positive and responsive to tamoxifen. The authors studied 12 mucinous carcinomas of the skin and compared the morphology with that of typical mammary lesions. The authors also evaluated for expression of estrogen receptor, progesterone receptor, and the mucus-associated peptides of the trefoil factor family (TFF), TFF1 (formerly pS2) and TFF2 (formerly SP), using immunohistochemistry. The localization of mRNAs for TFF1, TFF2, and TFF3 (formally ITF) was also studied in a subset of three tumors, using in-situ hybridization with S35 labeled riboprobes. The Grimelius stain was used to look for evidence of neuroendocrine differentiation. Eight resembled type A mucinous carcinomas of the breast, two resembled type B, and one had composite features. The 12th was a papillary neoplasm. The two type B tumors exhibited argyrophilia. All showed strong nuclear staining with the estrogen receptor antibody but a more varied pattern with antibodies to progesterone receptor and TFF1. None labeled for TFF2. The detection of TFF1 in mammalian skin is a novel finding. Cutaneous mucinous carcinoma shows strong similarities to its mammary counterpart, including expression of estrogen receptor, TFF1, and TFF3 mRNA. These observations suggest that some mucinous carcinomas of the skin might respond to antiestrogenic therapies.

Inhibition of hexonate dehydrogenase and aldose reductase from bovine retina by sorbinil, statil, M79175 and valproate.

Aldose reductase inhibitors (A.R.I.s), developed as potentially therapeutic agents for the treatment of complications of long-term diabetes, were found to be potent inhibitors of aldose reductase (ALR2) partially purified from bovine retina (IC50 values: Statil 0.89 microM, Sorbinil 2 microM, M79175 greater than 1 microM). These compounds varied, however, in their ability to inhibit hexonate dehydrogenase (ALR1), a closely related enzyme isolated from the same source (IC50 values: Statil greater than 1 microM, Sorbinil 3.9 microM, M79175 0.18 microM). Statil and Sorbinil were active against ALR2 at very low concentrations (approx. 5% inhibition at 100 pM), but did not inhibit ALR1 at less than or equal to 10 nM. In contrast, M79175 (structurally very similar to Sorbinil) and M7HEQ (a flavonoid) were preferential inhibitors of ALR1. Valproate, a compound of value in the treatment of epilepsies, was a poor inhibitor of ALR2 (18% at 1 mM). Furthermore, valproate was found to be a relatively poor inhibitor of ALR1, particularly in comparison with M79175.

Comparison of aldose reductase inhibitors in vitro. Effects of enzyme purification and substrate type.

Aldose reductase (EC 1.1.1.21) was purified approximately 5000-fold from bovine lens by ammonium sulphate fractionation and chromatography on DEAE-Sephacel and Matrex OA. Inhibition of this enzyme was found to depend upon the assay substrate. Tested against the purest form of enzyme, the inhibitor Sorbinil gave IC50 values of approximately 100 microM with the model substrate 4-nitrobenzaldehyde (4NB) and 0.4-1.4 microM with the physiological substrate glucose. A similar effect of substrate was found for the inhibitor Statil (IC50 450-750 nM with 4NB, 26-71 nM with glucose substrate). The implications of these results towards the assessment of aldose reductase inhibitors in vitro are discussed.

Inhibition of aldose reductase in five tissues of the streptozotocin-diabetic rat.

For 22 days, streptozotocin-diabetic and normal rats were intubated once daily with ICI 105552 (1-(3,4-dichlorobenzyl)-3-methyl-1,2-dihydro-2-oxoquinol-4-ylacetic acid, sodium salt: 50 mg/kg body weight) an inhibitor of aldose reductase (EC 1.1.1.21), the first enzyme of the sorbitol pathway. Treatment with ICI 105552 affected neither glycaemia nor tissue glucose nor inositol concentrations yet reduced significantly the abnormal accumulations in diabetes of sorbitol in the lens (70% reduction), sciatic nerve (86%) and seminal vesicles with coagulating glands (S.V.C.G., 55%). ICI 105552 had no effect upon sorbitol accumulated in the diabetic kidney but it reduced the level in controls by 43%. The compound reduced the accumulation of sorbitol in diabetic retina by 58% although variation was too great for the decrease to be significant statistically. Treatment with ICI 105552 produced small (less than or equal to 11%) yet statistically significant increases in the weights of the kidneys, and both liver and kidney weight/100 g residual body weight but did not affect the weights of the body, lens, retina or S.V.C.G. The importance of these findings for the development of potentially chemotherapeutic aldose reductase inhibitors is discussed.

The expression of E-cadherin and catenins in sporadic colorectal carcinoma.

The E-cadherin/catenin complex plays a major role in epithelial cell-cell adhesion. Immunohistochemical studies have highlighted perturbation in the expression and distribution of E-cadherin and catenins in sporadic colorectal neoplasms. In this study, we compared the expression of E-cadherin and catenins (alpha-, beta-, and gamma-catenin) in 30 sporadic colorectal carcinomas with that in the adjacent nonneoplastic mucosa and assessed whether any perturbation in the level of expression occurred at the messenger RNA (mRNA) or protein level. We also compared the expression of E-cadherin and catenins in 13 lymph node deposits and the primary tumors. Immunohistochemistry was used to study the level of expression and cellular distribution of E-cadherin and catenins. Levels of mRNA were studied by in situ hybridization. E-cadherin and catenin immunoreactivity was increased with cytoplasmic accumulation in more than 85% of the neoplasms. There were marked increases in the levels of mRNA in the carcinomas compared with the nonneoplastic mucosa. Nuclear localization of beta-catenin was higher at the invasive margin of some tumors, but expression of E-cadherin and catenin transcripts in the lymph node deposits showed no consistent relationship to that in the primary tumors.

Trefoil factor expression in normal and diseased human salivary glands.

Trefoil factors are wound-healing peptides important in protection and healing of the human gastrointestinal tract. Their potential for therapy of gastrointestinal ulcers has been established. This study investigated the hypothesis that trefoil factors are also present in human salivary gland. Tissues from surgical biopsy specimens were collected fresh into ice and stored in liquid nitrogen. Breast, stomach, and colon constituted positive controls. Trefoil factor mRNAs were detected by reverse transcription polymerase chain reaction (RT-PCR) or by in situ hybridization (ISH) with formalin-fixed, paraffin-embedded sections. Amplified DNA fragments were ligated into pGEM-T Easy vector and used to transform competent Escherichia coli JM109, allowing sequencing to confirm identity of cloned fragments. Generation of amplifiable cDNA was confirmed using primers specific to the ubiquitously expressed abl gene. By RT-PCR, TFF1 (pS2) mRNA was detected in 14 of 15 glands, TFF3 (hITF) mRNA in 13, and TFF2 (hSP) in only 1 gland. ISH of 15 glands (7 of which had been studied by RT-PCR) showed the same pattern of expression and indicated that TFF1 mRNA was usually expressed at low levels by a few mucous cells, whereas TFF3 was produced abundantly by most mucous cells. There was no difference in patterns of expression comparing parotid, submandibular, and minor mucous glands. Nor was there an obvious relationship between trefoil factor expression and pathology, but those glands not expressing TFF1 or TFF3 had evidence of chronic inflammation or atrophy. Trefoil factors are likely to be important in healing, predisposition to, and therapy of, oral diseases.

MT1-MMP and MMP-2 mRNA expression in human ovarian tumors: possible implications for the role of desmoplastic fibroblasts.

Expression of activated MMP-2 (72 kDa type IV collagenase) is highly associated with the malignant phenotype in adenocarcinomas, but predominant expression of the mRNA appears to be in stromal cells. MT1-MMP (membrane type 1-matrix metalloproteinase) is implicated in tumor-epithelial cell surface activation of latent pro-MMP-2, indicating a mechanism for tumor-stromal interaction in invasion. We determined the relative mRNA distribution of these MMPs in human ovarian tumors with a view to analyzing potential variations in the epithelial-mesenchymal interactions dictating ovarian tumor cell spread. In situ hybridization using 35S-labeled riboprobes was used to analyze 33 human ovarian tumors and mouse xenografts of human ovarian (DOV 13, SKOV3) and breast (MCF 7) tumor cell lines known to express MT1-MMP and MMP-2. MMP-2 mRNA was expressed in 31 of 33 and MT1-MMP mRNA was expressed in 29 of 33 tumor cases. MMP-2 mRNA was predominantly expressed in desmoplastic fibroblasts and in the subepithelial stroma. MT1-MMP mRNA showed some colocalization with MMP-2 in stromal cells. Neoplastic epithelial cell labeling for MT1-MMP mRNA was present in borderline and malignant tumors but not in benign tumors, and was invariably less than stromal labeling. Xenografts of DOV 13, SKOV 3, and MCF 7 cells showed some stromal localization of MMP-2 mRNA and weak labeling of DOV 13 cells. There was variable labeling for MT1-MMP mRNA in the neoplastic cells only. The colocalization of MT1-MMP and MMP-2 mRNAs in ovarian carcinoma stroma supports the view that MT1-MMP is closely associated with MMP-2 expression and function. It suggests that either additional mechanisms are involved in regulating MMP-2 activation at the tumor cell surface, or more intriguingly, that desmoplastic fibroblasts may be the primary mediators of extracellular matrix remodeling with respect to this system.

Expression of gelatinase A and TIMP-2 mRNAs in desmoplastic fibroblasts in both mammary carcinomas and basal cell carcinomas of the skin.

AIMS: To compare the localisation of mRNAs for the basement membrane degrading enzyme gelatinase A (72 kilodalton type IV collagenase) and its inhibitor TIMP-2 in carcinomas of the breast and basal cell carcinomas of the skin which have little or no ability to metastasize. METHODS: In situ hybridisation was performed on formalin fixed, paraffin wax embedded blocks using 35S-labelled riboprobes on 16 mammary carcinomas, three fibroadenomas, and a benign phyllodes tumour, and on 15 basal cell carcinomas of the skin (BCC). RESULTS: Labelling for both mRNAs was detectable in 14 of 16 mammary carcinomas and in 13 of 15 BCC, most often over organising desmoplastic fibroblasts in the stroma around invasive epithelial aggregates. Some sparse labelling was seen over malignant epithelial cells in six of the mammary carcinomas but not in the BCC. Some expression of gelatinase A mRNA was also seen in fibroblasts of breast lobules adjacent to the mammary carcinomas and around engulfed adnexal elements in the BCC, but not in unaffected breast tissues, fibroadenomas, the phyllodes tumour or unaffected skin. CONCLUSIONS: Maximal expression of gelatinase A and TIMP-2 mRNAs occurs in malignant neoplasms as part of the host response to the presence of established neoplastic cells rather than as an initial response to invasion. The degree to which this is present suggests this may be a highly relevant mechanism modulating tumour differentiation, growth and progression, possibly entailing uptake via specific receptors on the tumour cell surface.