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1.
Rev Sci Instrum ; 82(3): 034301, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21456768

ABSTRACT

A high performance gel imaging system was constructed using a digital single lens reflex camera with epi-illumination to image 19 × 23 cm agarose gels with up to 10,000 DNA bands each. It was found to give equivalent performance to a laser scanner in this high throughput DNA fingerprinting application using the fluorophore SYBR Green(®). The specificity and sensitivity of the imager and scanner were within 1% using the same band identification software. Low and high cost color filters were also compared and it was found that with care, good results could be obtained with inexpensive dyed acrylic filters in combination with more costly dielectric interference filters, but that very poor combinations were also possible. Methods for determining resolution, dynamic range, and optical efficiency for imagers are also proposed to facilitate comparison between systems.


Subject(s)
Lenses , Optical Phenomena , Sepharose/chemistry , Chromosomes, Artificial, Bacterial/genetics , DNA Fingerprinting , Gels , Lenses/economics , Limit of Detection
2.
J Endocrinol ; 177(1): 65-81, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12697038

ABSTRACT

Keratinocyte growth factor/fibroblast growth factor 7 (KGF/FGF7) is known to be a potent growth factor for mammary cells but its origin, cellular targets and mode of action in the breast are unclear. In this study, we carried out studies to determine the localisation of FGF7 and its receptor, and the related growth factor FGF10. We also determined the factors that regulate FGF7 release from stromal cells and the effects of FGF7 on normal and neoplastic breast cells. Using an FGF7-specific antibody which does not react with the FGF7 heparan sulphate proteoglycan (HSPG)-binding site, we showed epithelial and myoepithelial immunohistochemical staining in normal breast sections, and epithelial staining in breast carcinomas. Stromal staining was also detected in some lobular carcinomas as well as a subset of invasive ductal carcinomas. FGF10 and FGF receptor (FGFR)2 immunostaining showed a similar epithelial expression pattern, whereas no stromal staining was observed. We purified normal breast stromal, epithelial and myoepithelial cells and showed that FGF7 stimulated proliferation of both epithelial cell types, but not stromal fibroblasts. We also examined the effects of FGF7 on Matrigel-embedded organoids, containing both epithelial and myoepithelial cells, and showed FGF7 induced an increase in cellular proliferation. Furthermore, conditioned medium derived from stromal cells was shown to increase the proliferation of normal and neoplastic breast epithelial cells, which could be abolished by a neutralising antibody to FGF7. Finally, we showed that interleukin-1beta, but not oestradiol or other oestrogen receptor ligands, caused a dose-related FGF7 release. Further results also indicate that the epithelial localisation of FGF7 and FGF10 in breast tissue sections is likely to be due to their binding to their cognate receptor. In summary, our findings suggest that FGF7 is a paracrine growth factor in the breast. FGF7 is produced by the breast stromal fibroblasts and has profound proliferative and morphogenic roles on both epithelial and myoepithelial cells.


Subject(s)
Breast Neoplasms/metabolism , Breast/metabolism , Fibroblast Growth Factors/analysis , Interleukin-1/pharmacology , Paracrine Communication/physiology , Blotting, Western/methods , Breast Neoplasms/chemistry , Carcinoma, Ductal, Breast/chemistry , Carcinoma, Lobular/chemistry , Cell Division/drug effects , Cells, Cultured , Enzyme-Linked Immunosorbent Assay/methods , Epithelial Cells/chemistry , Epithelial Cells/cytology , Epithelial Cells/drug effects , Female , Fibroblast Growth Factor 10 , Fibroblast Growth Factor 7 , Fibroblast Growth Factors/metabolism , Fibroblast Growth Factors/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Receptor Protein-Tyrosine Kinases/analysis , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 2 , Receptors, Fibroblast Growth Factor/analysis , Receptors, Fibroblast Growth Factor/metabolism , Tumor Cells, Cultured
3.
Biotechniques ; 33(3): 532-4, 536, 538-9, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12238763

ABSTRACT

We are investigating approaches to increase DNA sequencing quality. Since a majorfactor in sequence generation is the cost of reagents and sample preparations, we have developed and optimized methods to sequence directly plasmid DNA isolated from alkaline lysis preparations. These methods remove the costly PCR and post-sequencing purification steps but can result in low sequence quality when using standard resuspension protocols on some sequencing platforms. This work outlines a simple, robust, and inexpensive resuspension protocol for DNA sequencing to correct this shortcoming. Resuspending the sequenced products in agarose before electrophoresis results in a substantial and reproducible increase in sequence quality and read length over resuspension in deionized water and has allowed us to use the aforementioned sample preparation methods to cut considerably the overall sequencing costs without sacrificing sequence quality. We demonstrate that resuspension of unpurified sequence products generated from template DNA isolated by a modified alkaline lysis technique in low concentrations of agarose yields a 384% improvement in sequence quality compared to resuspension in deionized water. Utilizing this protocol, we have produced more than 74,000 high-quality, long-read-length sequences from plasmid DNA template on the MegaBACET 1000 platform.


Subject(s)
DNA, Bacterial/genetics , Plasmids/genetics , Sepharose , Sequence Analysis, DNA/instrumentation , Sequence Analysis, DNA/methods , Cell Fractionation/methods , DNA, Bacterial/isolation & purification , Electrophoresis, Agar Gel/instrumentation , Electrophoresis, Agar Gel/methods , Plasmids/isolation & purification , Quality Control , Reproducibility of Results , Sensitivity and Specificity
4.
Br J Cancer ; 86(7): 1097-103, 2002 Apr 08.
Article in English | MEDLINE | ID: mdl-11953856

ABSTRACT

Fibroblast growth factor 8 can transform NIH3T3 cells and its expression has been found to be associated with breast and prostate cancer. Following our finding that fibroblast growth factor 8 mRNA expression is increased in breast cancer, we have undertaken an immunohistochemistry study of fibroblast growth factor 8 expression in a series of human breast tissues and other normal tissues. Our findings confirm increased expression of fibroblast growth factor 8 in malignant breast tissue but also show significant fibroblast growth factor 8 expression in non-malignant breast epithelial cells. No significant difference in fibroblast growth factor 8 expression was found between different grades of ductal carcinoma, lobular carcinoma and ductal carcinoma in-situ or cancer of different oestrogen receptor, progesterone receptor or nodal status. The highest levels of fibroblast growth factor 8 expression were found in lactating breast tissues and fibroblast growth factor 8 was also detected in human milk. A survey of other normal tissues showed that fibroblast growth factor 8 is expressed in the proliferative cells of the dermis and epithelial cells in colon, ovary fallopian tube and uterus. Fibroblast growth factor 8 appears to be expressed in several organs in man and appears to have an importance in lactation.


Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/pathology , Fibroblast Growth Factors/biosynthesis , Gene Expression Regulation, Neoplastic , Lactation , Adult , Aged , Aged, 80 and over , Cell Division , Colon/physiology , Epithelial Cells/physiology , Fallopian Tubes/physiology , Female , Fibroblast Growth Factor 8 , Humans , Immunohistochemistry , Middle Aged , Milk, Human/chemistry , Uterus/physiology
5.
J Pathol ; 194(1): 27-34, 2001 May.
Article in English | MEDLINE | ID: mdl-11329138

ABSTRACT

Immunohistochemical staining of human breast tissues, using an antibody against fibroblast growth factor receptor 3 [FGFR-3], showed differences in cellular distribution. Both malignant and non-malignant epithelial cells contained FGFR-3 immunoreactivity, but myoepithelial cells and stroma were negative. The staining pattern in malignant epithelial cells was predominantly nuclear, whereas epithelial cells in normal breast tissue showed both cytoplasmic and nuclear elements. Reverse transcription-polymerase chain reaction (RT-PCR) revealed two isoforms of FGFR-3 corresponding to the FGFR-3-IIIb variant and a previously described exon-deleted nuclear form of FGFR-3, which were present in both malignant and non-malignant epithelial cells. The higher level of nuclear staining and loss of cytoplasmic staining seen in malignant epithelial cells did not correspond to an increase in expression of the exon-deleted form of FGFR-3, nor to any detectable activating point mutations. Since receptor activation can result in its movement to a perinuclear localization, an alternative explanation for the redistribution of FGFR-3-IIIb could be different degrees of activation by a ligand (FGF1 or FGF9). No FGF9 was detected by immunohistochemistry in breast tissues. FGF1, however, is present in the majority of breast cancers and a different tissue distribution of FGF1 was found in breast tissues, showing predominantly nuclear, or a mix of nuclear and cytoplasmic FGFR-3. The difference in FGFR-3 staining patterns may implicate this ligand-receptor pair in breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Neoplasm Proteins/metabolism , Protein-Tyrosine Kinases , Receptors, Fibroblast Growth Factor/metabolism , Adult , Aged , Aged, 80 and over , Cell Nucleus/metabolism , Female , Fibroblast Growth Factor 1 , Fibroblast Growth Factor 2/metabolism , Fibroblast Growth Factors/metabolism , Gene Expression , Humans , Immunoenzyme Techniques , Middle Aged , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Receptor, Fibroblast Growth Factor, Type 3 , Receptors, Fibroblast Growth Factor/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
6.
Oncogene ; 18(4): 1053-60, 1999 Jan 28.
Article in English | MEDLINE | ID: mdl-10023681

ABSTRACT

Fibroblast growth factor 8 (FGF8) is an important developmental protein which is oncogenic and able to cooperate with wnt-1 to produce mouse mammary carcinoma. The level of expression of FGF8 mRNA was measured in 68 breast cancers and 24 non-malignant breast tissues. Elevated levels of FGF8 mRNA were found in malignant compared to non-malignant breast tissues with significantly more malignant tissues expressing FGF8 (P=0.019) at significantly higher levels (P=0.031). In situ hybridization of breast cancer tissues and analysis of purified populations of normal epithelial cells and breast cancer cell lines showed that malignant epithelial cells expressed FGF8 mRNA at high levels compared to non-malignant epithelial and myoepithelial cells and fibroblasts. Although two of the receptors which FGF8 binds to (FGFR2-IIIc, FGFR3-IIIc) are not expressed in breast cancer cells, an autocrine activation loop is possible since expression of fibroblast growth factor receptor (FGFR) 4 and FGFR1 are retained in malignant epithelial cells. This is the first member of the FGF family to have increased expression in breast cancer and a potential autocrine role in its progression.


Subject(s)
Breast Neoplasms/metabolism , Breast/metabolism , Fibroblast Growth Factors/metabolism , Neoplasm Proteins/metabolism , Protein-Tyrosine Kinases , Receptors, Fibroblast Growth Factor/metabolism , Adult , Aged , Epithelial Cells/metabolism , Female , Fibroblast Growth Factor 8 , Humans , In Situ Hybridization , Middle Aged , RNA, Messenger/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 1 , Receptor, Fibroblast Growth Factor, Type 3 , Receptor, Fibroblast Growth Factor, Type 4 , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
7.
Exp Cell Res ; 247(1): 267-78, 1999 Feb 25.
Article in English | MEDLINE | ID: mdl-10047469

ABSTRACT

We show that myoepithelial cell basement membrane derived E3 and E8 domains of laminin-1 are capable of polarizing luminal epithelial cells with regard to epithelial membrane antigen localization. This event is dependent on the alpha6 integrin and results in aggregation and phosphorylation of the tyrosine residues of the focal adhesion kinase complex. We also demonstrate that uncultured normal luminal epithelial cells synthesize normal levels of beta and gamma laminin chains and reduced levels of alpha chains mRNA in common with malignant epithelial cells. In contrast normal myoepithelial cells synthesize all three constituent chains of laminin-1. Therefore in breast cancer the absence of myoepithelial cells could result in a lack of laminin alpha chains which may contribute to loss of polarity of malignant epithelial cells.


Subject(s)
Breast/cytology , Cell Polarity/physiology , Epithelial Cells/physiology , Amino Acid Sequence , Basement Membrane/pathology , Basement Membrane/physiology , Breast/pathology , Breast/physiology , Breast Neoplasms , Cell Communication/physiology , Cell Line , Cells, Cultured , Epithelial Cells/metabolism , Female , Humans , Laminin/biosynthesis , Molecular Sequence Data , Tumor Cells, Cultured
8.
Exp Cell Res ; 234(1): 165-73, 1997 Jul 10.
Article in English | MEDLINE | ID: mdl-9223382

ABSTRACT

We have studied separated normal human breast epithelial and myoepithelial cells for the presence of basic fibroblast growth factor (FGF2) and its receptors, both low (heparan sulfate proteoglycans) and high affinity (FGFR1), and for the effects of FGF2 on the proliferation of both cell types. Our results indicate that these cells differ markedly in their synthesis and response to FGF2. We found, using PCR of purified cell populations, mRNA for FGF2 only in the myoepithelial cells, whereas immunostaining and Western blotting results demonstrated the presence of FGF2 protein in both epithelial and myoepithelial cells. FGF2 had no effect on the proliferation of myoepithelial cells, but it did maintain the survival of the separated epithelial cells in low serum and stimulate their growth in 5% and 10% FCS. Immunostainable FGFR1 was present in epithelial cells and, to a lesser extent, in myoepithelial cells. Low-affinity binding sites for FGF2 were synthesized by epithelial and myoepithelial cells, but myoepithelial cells possessed a greater proportion of higher-affinity heparan sulfate proteoglycans. These results indicate that myoepithelial cell-derived FGF2 may be an important paracrine factor controlling epithelial cell survival and growth in the normal human breast.


Subject(s)
Breast/cytology , Fibroblast Growth Factor 2/physiology , Receptor Protein-Tyrosine Kinases/analysis , Receptors, Fibroblast Growth Factor/analysis , Blotting, Southern , Blotting, Western , Cell Division/physiology , Cells, Cultured , Chromatography, High Pressure Liquid , DNA Primers , Epithelial Cells , Epithelium/chemistry , Epithelium/metabolism , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Female , Fibroblast Growth Factor 2/biosynthesis , Fibroblast Growth Factor 2/genetics , Glucosamine/analysis , Humans , Immunohistochemistry , Mitogens/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Fibroblast Growth Factor, Type 2 , Receptors, Fibroblast Growth Factor/genetics , Subcellular Fractions/chemistry , Tetrazolium Salts , Tritium
9.
J Cell Physiol ; 171(1): 11-9, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9119887

ABSTRACT

In order to investigate the specific factors controlling the growth of normal breast cell types, purified populations of human breast epithelial and myoepithelial cells from reduction mammoplasties were grown in primary culture in three defined media and their response to foetal calf serum (FCS), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) measured using MTT growth assays. Epithelial and myoepithelial cells differed markedly in their growth requirements. Whereas epithelial cell survival was dependent on the presence of FCS, myoepithelial cell growth was dramatically inhibited by serum. EGF and FGF2 were mitogenic for epithelial cells but not myoepithelial cells, the addition of insulin being the only essential supplement required for myoepithelial cell growth. Heparin inhibited FGF2-stimulated epithelial cell growth but also basal myoepithelial cell proliferation and this inhibition could be overcome by the addition of EGF. Neutralizing antibodies to EGF also inhibited basal myoepithelial cell growth. This suggests the possibility of an autocrine role for a heparin-binding member of the EGF family in the growth of myoepithelial cells. Purified cells combined to form lobuloalveolar structures when incubated in a reconstituted basement membrane matrix (Matrigel) in the presence of EGF and FGF2.


Subject(s)
Breast/cytology , Epidermal Growth Factor/pharmacology , Fibroblast Growth Factor 2/pharmacology , Animals , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Separation , Cells, Cultured , Epithelial Cells , Female , Humans , Rats
10.
Br J Cancer ; 75(11): 1621-30, 1997.
Article in English | MEDLINE | ID: mdl-9184178

ABSTRACT

Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 37 degrees C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast.


Subject(s)
Breast Neoplasms/chemistry , Endopeptidases/metabolism , Fibroblast Growth Factor 1/analysis , Receptor Protein-Tyrosine Kinases , Animals , Female , Humans , Immunoblotting , Immunohistochemistry , Mice , Polymerase Chain Reaction , Receptor, Fibroblast Growth Factor, Type 1 , Receptor, Fibroblast Growth Factor, Type 4 , Receptors, Fibroblast Growth Factor/analysis
11.
Br J Cancer ; 75(8): 1173-84, 1997.
Article in English | MEDLINE | ID: mdl-9099967

ABSTRACT

A variant form of the human oestrogen receptor (ER) mRNA lacking sequences encoded within exon 5 has been described (Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O'Malloy BW, McGuire WL 1991, Cancer Res 51: 105-109). We have examined the expression of the exon 5-deleted ER (HE delta5) mRNA variant in breast biopsies using reverse transcriptase polymerase chain reaction (RT - PCR). HE delta5 mRNA was present in only 13% of non-malignant breast tissues compared with 32% of carcinomas (95% CI, P=0.05). Presence of the HE delta5 mRNA was associated with the presence of immunohistochemically detected ER (P=0.015) and progesterone receptor (PR) (P=0.02). There was a positive correlation between the presence of HE delta5 and disease-free survival (P=0.05), suggesting that the presence of HE delta5 may be an indicator of better prognosis. We have raised a monoclonal antibody specific to the C-terminal amino acids of HE delta5. This antibody recognized the variant but not the wild-type ER protein. We show that HE delta5 protein is present in breast cancer using immunohistochemical techniques. We also analysed trans-activation by HE delta5 in mammalian cells and showed that, in MCF-7 cells, HE delta5 competes with wild-type ER to inhibit ERE-dependent trans-activation. Our results indicate that this variant is unlikely to be responsible for endocrine resistance of breast cancer, but its presence at both the mRNA and protein level suggest that it may, nevertheless, be involved in regulating the expression of oestrogen-responsive genes in breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Exons , Gene Expression Regulation, Neoplastic , Receptors, Estrogen/biosynthesis , Sequence Deletion/genetics , Adult , Aged , Aged, 80 and over , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/biosynthesis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , COS Cells , DNA Primers/chemistry , Enzyme-Linked Immunosorbent Assay , Female , Humans , Mice , Mice, Inbred BALB C , Middle Aged , RNA, Messenger/biosynthesis , Receptors, Estrogen/genetics , Receptors, Estrogen/immunology , Receptors, Progesterone/biosynthesis , Survival Analysis , Transfection/genetics , Tumor Cells, Cultured
12.
Br J Cancer ; 75(1): 28-33, 1997.
Article in English | MEDLINE | ID: mdl-9000594

ABSTRACT

This paper examines the expression of fibroblast growth factor 2 (FGF-2) in the malignant human breast. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the level of expression of FGF-2 in a series of 51 patients clinically followed up for a median of 84 months (Luqmani et al, 1992). Immunohistochemistry and Western blotting were used to show that the level of FGF-2 in breast tissues correlated with the amount of FGF-2 mRNA. FGF-2 was present in both malignant and non-malignant breast, although less was expressed in malignant tissues as determined by all three methods. Immunohistochemistry on frozen sections of breast tissue showed expression of FGF-2 in myoepithelial and epithelial cells in non-malignant samples and generally lower or undetectable levels of staining in malignant epithelial cells. The results obtained by immunohistochemistry correlated well with RT-PCR data showing similar levels of FGF-2 and FGF-2 mRNA expression in samples. No correlation was found between FGF-2 mRNA expression and T stage, nodal status or oestrogen receptor status. However, Kaplan-Meier survival plots show that higher levels of FGF-2 are associated with improved overall and disease-free survival. We suggest that FGF-2 expression may have value as a prognostic indicator in breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Carcinoma/metabolism , Fibroblast Growth Factor 2/biosynthesis , Adult , Aged , Blotting, Western , Breast/chemistry , Breast/metabolism , Breast Neoplasms/chemistry , Breast Neoplasms/diagnosis , Carcinoma/chemistry , Carcinoma/diagnosis , Carcinoma, Ductal, Breast/chemistry , Carcinoma, Ductal, Breast/diagnosis , Disease-Free Survival , Female , Fibroblast Growth Factor 2/analysis , Humans , Immunohistochemistry , Middle Aged , Prognosis , RNA, Messenger/analysis , RNA, Neoplasm/chemistry , Survival Analysis
13.
Br J Cancer ; 76(11): 1419-27, 1997.
Article in English | MEDLINE | ID: mdl-9400937

ABSTRACT

Monoclonal antibodies against two epitopes of FGFR-1 have been used to investigate FGFR-1 expression in the normal and neoplastic human breast. Different forms are detected in the different cell types constituting the normal breast. Moreover, breast cancer cells lack one form of FGFR-1. Western blot analysis showed 115-kDa and 106-kDa forms of FGFR-1 within the human breast. The 115-kDa band corresponds to the beta form of FGFR-1, whereas the 106-kDa band is truncated at the carboxyl terminus. The 106-kDa form of FGFR-1 is the major form present in breast fibroblasts and myoepithelial cells, whereas epithelial cells contain equal amounts of the 115-kDa and 106-kDa forms. Breast cancer cells, however, appear to contain only the 115-kDa form of FGFR-1. This expression pattern is reflected in malignant and non-malignant tissue samples. Using reverse transcription polymerase chain reaction (RT-PCR) analysis, we have shown that the 106-kDa FGFR-1 isoform is not the previously described alpha 2 receptor that arises from a 25-base pair insertion in the second kinase domain. It is probable that the 106-kDa FGFR-1 has different signalling properties to the full-length receptor, having lost at least one tyrosine at amino acid 766, which is required for phospholipase C activation. This form of FGFR-1 appears to be lost in all breast cancer cells analysed and its absence may have a bearing on malignancy.


Subject(s)
Breast Neoplasms/ultrastructure , Receptor Protein-Tyrosine Kinases , Receptors, Fibroblast Growth Factor/metabolism , Amino Acid Sequence , Antibodies, Monoclonal , Breast/cytology , Breast/metabolism , Breast/ultrastructure , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Down-Regulation/physiology , Epithelial Cells/ultrastructure , Fibroblasts/ultrastructure , Humans , Immunohistochemistry , Isomerism , Molecular Sequence Data , Molecular Weight , RNA, Messenger/metabolism , Receptor, Fibroblast Growth Factor, Type 1 , Receptors, Fibroblast Growth Factor/biosynthesis , Receptors, Fibroblast Growth Factor/immunology , Tumor Cells, Cultured
14.
Oncol Rep ; 4(2): 425-8, 1997.
Article in English | MEDLINE | ID: mdl-21590072

ABSTRACT

Fibroblast growth factor receptors are widely expressed on breast cancer cells and we report a preliminary study to determine whether these could be useful as potential targets for delivery of a cytotoxic fibroblast growth factor 2-saporin conjugate. We show that this mitotoxin conjugate can displace I-125-fibroblast growth factor 2 binding though with reduced affinity compared to unlabeled fibroblast growth factor 2. For 4 out of 5 cell lines it is an effective inhibitor of cell growth, and cytotoxic for at least 2 of the lines. Inhibitory effects did not depend on responsiveness of cells to fibroblast growth factor 2. This activity was not achieved with free saporin. There may be potential uses for this conjugate in both experimental systems to study receptor function and subsequent processing, and also in clinical settings to eliminate breast cancer cells.

15.
Br J Cancer ; 72(6): 1420-6, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8519654

ABSTRACT

We have measured the amount of fibroblast growth factor 1 (FGF-1) mRNA and protein in primary breast cancers and non-malignant breast tissue and have found greatly reduced levels in breast cancer compared with non-malignant tissue. A total of 116 breast cancers and 37 biopsies taken from non-malignant breast were compared for FGF-1 mRNA expression using reverse transcriptase-polymerase chain reaction (RT-PCR) and significantly lower levels were found in the cancer tissues (P < 0.001). These findings were confirmed at the protein level where four out of five breast cancers contained no detectable FGF-1 and a fifth cancer had a low level of FGF-1 compared with three samples from reduction mammoplasties. Similar results were obtained from breast cell lines in which 80% of cancer cell lines had very low levels of FGF-1, whereas all non-malignant breast cell lines contained higher levels of FGF-1. Immunohistochemical analysis indicated that FGF-1 was present in the luminal epithelial cells of the non-malignant breast but was absent from cancer cells. The decreased levels of FGF-1 in breast cancer may indicate that stimulation of cancer cells is resulting in down-regulation of FGF-1 expression or may implicate FGF-1 as a differentiation factor rather than a growth factor at its physiological concentration in the breast.


Subject(s)
Breast Neoplasms/metabolism , Breast/metabolism , Fibroblast Growth Factor 1/biosynthesis , Adult , Aged , Aged, 80 and over , Base Sequence , Blotting, Western , Breast/chemistry , Breast Neoplasms/chemistry , Cell Line , Female , Fibroblast Growth Factor 1/analysis , Humans , Immunohistochemistry , Middle Aged , Molecular Sequence Data , Neoplasm Proteins/analysis , Neoplasm Proteins/biosynthesis , Protein Biosynthesis , Proteins/analysis , RNA, Messenger/analysis , RNA, Messenger/metabolism , Receptors, Fibroblast Growth Factor/genetics , Reference Values , Tumor Cells, Cultured
16.
Anal Biochem ; 226(1): 91-9, 1995 Mar 20.
Article in English | MEDLINE | ID: mdl-7785785

ABSTRACT

Monoclonal antibodies to epithelial membrane antigen and common acute lymphoblastic leukemia antigen were bound to second antibody-coated magnetic microspheres. These specific antibody/bead complexes were then used to directly isolate purified epithelial and myoepithelial cells from normal breast organoid cell preparations by magnetic separation. Near homogeneous cell populations were selected with yields of 10-20 x 10(6) epithelial and myoepithelial cells per organoid preparation. Repeated purification steps allowed almost complete depletion of myoepithelial cells for RNA studies. Tissue culture of separated cell populations in appropriate defined media further ensured purity of cell type and was unimpeded by Dynabead attachment.


Subject(s)
Breast/cytology , Cell Separation/methods , Immunomagnetic Separation , Antibodies, Monoclonal/immunology , Base Sequence , Cells, Cultured , DNA, Complementary/genetics , Electrophoresis, Agar Gel , Epithelial Cells , Female , Gene Expression/genetics , Humans , Membrane Glycoproteins/immunology , Molecular Sequence Data , Mucin-1 , Mucins/immunology , Neprilysin/immunology
17.
Br J Cancer ; 69(5): 879-82, 1994 May.
Article in English | MEDLINE | ID: mdl-8180018

ABSTRACT

Limonene, a monocyclic monoterpene, occurs naturally in orange peel oil. It has been shown to exhibit both chemopreventive and chemotherapeutic activity without toxicity in rodent models. In this study we examined the effect of limonene both at maximally optimal and suboptimal doses and in combination with suboptimal doses of 4-hydroxyandrostrenedione on nitrosmethylurea-induced rat mammary tumours. A 10% limonene dose mixed in the diet caused tumour regression in all animals. A 5% limonene dose was only able to cause regression in 50% of the rats (P < 0.05). A suboptimal dose of 4-hydroxyandrostrenedione (12.5 mg kg-1) resulted in tumour regression in 75% of rats. A combination of 5% limonene with 4-hydroxyandrostrenedione (12.5 mg kg-1) resulted in a greater tumour regression (83.3%) than either agent given individually (P < 0.001 and 0.006 for limonene/4-hydroxyandrostrenedione vs limonene alone and 4-hydroxyandrostrenedione alone respectively.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Mammary Neoplasms, Experimental/drug therapy , Androstenedione/administration & dosage , Androstenedione/analogs & derivatives , Animals , Cyclohexenes , Drug Screening Assays, Antitumor , Female , Limonene , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/pathology , Methylnitrosourea , Rats , Rats, Inbred Strains , Terpenes/administration & dosage
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