In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

Background Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. Methods We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. Results In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. Conclusion We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.

Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

Background Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

An assessment of MMP and TIMP gene expression in cell lines and stroma : tumour differences in microdissected breast cancer biopsies

To examine matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) mRNA levels in archival breast cancer biopsies, we employed microdissection to separate tumour tissue from the surrounding breast tissue, or stroma and RT-PCR to determine gross qualitative and small quantitative differences in the patterns of expression. In this study, a significant correlation (p < 0.05, by Mann-Whitney U analysis) between TIMP-2 expression and lymph node involvement was identified, while MMP-11 and TIMP-1 expression patterning also significantly (p < 0.05) differed between those tumours showing calcification and those that did not. When compared by Spearmans’ ρ correlation analysis, a significant association (p < 0.05, ρ = 0.404) was identified in the pattern of MMP-2 and MMP-9 gene expression. In this study, the use of microdissection and a systematic strategy of RT-PCR analysis have allowed us to investigate localized MMP and MMP inhibitor expression within breast tumours. We have identified patterns of gene expression that may further reveal aspects of breast carcinogenesis, and a robust method for examining changes in clinically important genes using archival biopsies and across stroma-tumour boundaries.

Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis

To examine gene-expression patterning in late-stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD-PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor–stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma-associated genes were known. When applied to transformed breast cancer cell lines (MDA-MB-435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD-PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.

Chromosomal aberrations in squamous cell carcinoma and solar keratoses revealed by comparative genomic hybridization

OBJECTIVE: To identify chromosomal copy numbers of frequent genetic aberrations within squamous cell carcinomas (SCCs) and solar keratoses (SKs), and provide further evidence to support or challenge current dogma concerning the relationship between these lesions. DESIGN: Retrospective analysis of genetic aberrations in DNA from SK and SCC biopsy specimens by comparative genomic hybridization. SETTING: University-based research laboratory in Queensland, Australia. PATIENTS: Twenty-two biopsy specimens from patients with diagnosed SKs (n = 7), cutaneous SCCs (n = 10), or adjoining lesions (n = 5). MAIN OUTCOME MEASURES: Identification of frequent genetic aberrations both specific to SK and SCC and shared by these lesions to investigate their clonal relationship. RESULTS: Shared genomic imbalances were identified in SK and SCC. Frequent gains were located at chromosome arms 3q, 17q, 4p, 14q, Xq, 5p, 9q, 8q, 17p, and 20q, whereas shared regional losses were observed at 9p, 3p, 13q, 17p, 11p, 8q, and 18p. Significant loss of 18q was observed only in SCC lesions. CONCLUSIONS: Our results demonstrate that numerous chromosomal aberrations are shared by the 2 lesions, suggesting a clonal relationship between SK and SCC. Additionally, the genomic loss of 18q may be a significant event in SK progression to SCC. Finally, the type and frequency of aberrations suggests a common mode of tumorigenesis in SCC-derived tumors.

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell carcinoma and squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio = 3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information wained through observation of pigmentation phenotype.

Molecular cytogenetic analysis of basal cell carcinoma DNA using comparative genomic hybridization

In an attempt to define genomic copy number changes associated with the development of basal cell carcinoma, we investigated 15 sporadic tumors by comparative genomic hybridization. With the incorporation of tissue microdissection and degenerate oligonucleotide primed-polymerase chain reaction we were able to isolate, and then universally amplify, DNA from the tumor type. This combined approach allows the investigation of chromosomal imbalances within a histologically distinct region of tissue. Using comparative genomic hybridization we have observed novel and recurrent chromosomal gains at 6p (47%), 6q (20%), 9p (20%), 7 (13%), and X (13%). In addition comparative genomic hybridization revealed regional loss on 9q in 33% of tested tumors encompassing 9q22.3 to which the putative tumor suppressor gene, Patched, has been mapped. The deletion of Patched has been indicated in the development of hereditary and sporadic basal cell carcinomas. The identification of these recurrent genetic aberrations suggests that basal cell carcinomas may not be as genetically stable as previously thought. Further investigation of these regions may lead to the identification of other genes responsible for basal cell carcinoma formation.

Cytogenetics of basal cell carcinoma and squamous cell carcinomas

Cytogenetic analysis is a powerful tool that allows analysis of chromosomal aberrations associated with diseased states. In particular, a combination of cytogenetic techniques has allowed the identification of aberrations associated with cancer development, including cancers of the skin. This chapter provides a comprehensive overview of cytogenetic alterations in basal and squamous cell carcinomas of the skin. These two distinct lesions have altered karyotypes that are consistent with their malignant potential. Basal cell carcinomas, although relatively stable lesions, are highly associated with recurrent aberrations of chromosomes 6, 7, 9 and X, as detected by a number of cytogenetic techniques. Squamous cell carcinomas, on the other hand are associated with a much higher degree of instability, involving aberrations of chromosomes 3, 7, 8, 11, 13, 17 and 18, as detected using a number of cytogenetic techniques. Overall, the numbers and types of aberrations associated with basal and squamous cell carcinoma, define the characteristic behaviour associated with these lesions and identification of these aberrations may aid in the understanding of malignant potential, prognosis and treatment of these skin cancers.

A locus on the long arm of chromosome 1 as a possible cause of essential hypertension

None of the genes responsible for essential hypertension has been identified. Recent work in genetically hypertensive rats has shown linkage of blood pressure with alleles of the renin gene. Since the renin gene is a member of a conserved synteny group that in humans spans chromosome 1q21.3-32.3 and includes the gene for antithrombin III (AT3), we used linkage studies to examine the relationship between alleles of AT3 and hypertension in a family having 10 affected members. From the lod score obtained at a recombination fraction of zero the odds for linkage of AT3 and hypertension in this family were calculated as 6:1 in favour of linkage. This result provides grounds for further examination of the possible role of the 1q23 locus in the aetiology of essential hypertension.

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas

Background aims Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. Methods MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. Results Human L-MSC cultures were typically CD34−, CD45− and HLA-DR− and CD73+, CD90+, CD105+ and HLA-ABC+. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. Conclusions L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.

Low temperature pretreatment of sugarcane bagasse at atmospheric pressure using mixtures of ethylene carbonate and ethylene glycol

A low temperature lignocellulose pretreatment process was developed using acid-catalysed mixtures of alkylene carbonate and alkylene glycol. Pretreatment of sugarcane bagasse with mixtures of ethylene carbonate (EC) and ethylene glycol (EG) was more effective than that with mixtures of propylene carbonate (PC) and propylene glycol (PG). These mixtures were more effective than the individual components in making bagasse cellulose more amenable to cellulase digestion. Glucan digestibilities of ≥87% could be achieved with a wide range of EC to EG ratios from 9:1 to 1:1 (w/w). Pretreatment of bagasse by the EC/EG mixture with a ratio of 4:1 in the presence of 1.2% H2SO4 at 90 °C for 30 min led to the highest glucan enzymatic digestibility of 93%. The high glucan digestibilities obtained under these acidic conditions were due to (a) the ability of alkylene carbonate to cause significant biomass size reduction, (b) the ability of alkylene glycol to cause biomass defibrillation, (c) the ability of alkylene carbonate and alkylene glycol to remove xylan and lignin, and (d) the magnified above attributes in the mixtures of alkylene carbonate and alkylene glycol.

IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line

The in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) is a method that allows the direct localisation of gene expression. The method utilises the dual buffer mediated activity of the enzyme rTth DNA polymerase enabling both reverse transcription and DNA amplification. Labelled nucleoside triphosphates allow the site of expression to be labelled, rather than the PCR primers themselves, giving a more accurate localisation of transcript expression and decreased background than standard in situ hybridisation (ISH) assays. The MDA-MB-231 human breast carcinoma (HBC) cell line was assayed via the IS-RT-PCR technique, using primers encoding MT-MMP (membrane-type matrix metalloproteinase) and human β-actin. Our results clearly indicate baseline expression of MT-MMP in the relatively invasive MDA-MB-231 cell line at a signal intensity similar to the housekeeping gene β-actin, and results following induction with Concanavalin A (Con A) are consistent with our previous results obtained via Northern blotting.

Frequency in hypertensives of alleles for a RFLP associated with the renin gene

The genetic basis of primary hypertension is not known. Renin is important in blood pressure and volume control and a HindIII restriction fragment length polymorphism (RFLP) is present within the human renin gene locus. To examine whether there is a relationship between this RFLP and primary hypertension, DNA and renin analyses were performed on leukocytes and plasma from hypertensive and normotensive individuals. In hypertensives the frequencies of alleles for the HindIII RFLP were found to be 0.55 and 0.45, compared with 0.60 and 0.40 in the total population of 231 subjects examined, a difference that was not statistically significant. There also appeared to be no significant difference in renin activity in plasma for hypertensive patients of each genotype, nor in their pre- or post-treatment blood pressures. We thus conclude that, within the limits of the present study, the suspected genetic abnormalities associated with primary hypertension in man do not appear to be related to a HindIII RFLP in the renin gene.

Scanning the genome for essential hypertension loci

1. Essential hypertension occurs in people with an underlying genetic predisposition who subject themselves to adverse environmental influences. The number of genes involved is unknown, as is the extent to which each contributes to final blood pressure and the severity of the disease. 2. In the past, studies of potential candidate genes have been performed by association (case-control) analysis of unrelated individuals or linkage (pedigree or sibpair) analysis of families. These studies have resulted in several positive findings but, as one may expect, also an enormous number of negative results. 3. In order to uncover the major genetic loci for essential hypertension, it is proposed that scanning the genome systematically in 100- 200 affected sibships should prove successful. 4. This involves genotyping sets of hypertensive sibships to determine their complement of several hundred microsatellite polymorphisms. Those that are highly informative, by having a high heterozygosity, are most suitable. Also, the markers need to be spaced sufficiently evenly across the genome so as to ensure adequate coverage. 5. Tests are performed to determine increased segregation of alleles of each marker with hypertension. The analytical tools involve specialized statistical programs that can detect such differences. Non- parametric multipoint analysis is an appropriate approach. 6. In this way, loci for essential hypertension are beginning to emerge.