Biomedical subjects

L Happerfield

Publications and source records attributed to L Happerfield.

12 recordsLinked to original sources

PubMed2001

DNA replication licensing and human cell proliferation.

The convergence point of growth regulatory pathways that control cell proliferation is the initiation of genome replication, the core of which is the assembly of pre-replicative complexes resulting in chromatin being "licensed" for DNA replication in the subsequent S phase. We have analysed regulation of the pre-replicative complex proteins ORC, Cdc6, and MCM in cycling and non-proliferating quiescent, differentiated and replicative senescent human cells. Moreover, a human cell-free DNA replication system has been exploited to study the replicative capacity of nuclei and cytosolic extracts prepared from these cells. These studies demonstrate that downregulation of the Cdc6 and MCM constituents of the replication initiation pathway is a common downstream mechanism for loss of proliferative capacity in human cells. Furthermore, analysis of MCM protein expression in self-renewing, stable and permanent human tissues shows that the three classes of tissue have developed very different growth control strategies with respect to replication licensing. Notably, in breast tissue we found striking differences between the proportion of mammary acinar cells that express MCM proteins and those labelled with conventional proliferation markers, raising the intriguing possibility that progenitor cells of some tissues are held in a prolonged G1 phase or "in-cycle arrest". We conclude that biomarkers for replication-licensed cells detect, in addition to actively proliferating cells, cells with growth potential, a concept that has major implications for developmental and cancer biology.

Animals↗

PubMed1999

Macrophage-tumour cell interactions: identification of MUC1 on breast cancer cells as a potential counter-receptor for the macrophage-restricted receptor, sialoadhesin.

In many carcinomas, infiltrating macrophages are commonly found closely associated with tumour cells but little is known concerning the nature or significance of adhesion molecules involved in these cellular interactions. Here we demonstrate in primary human breast cancers that sialoadhesin (Sn), a macrophage-restricted adhesion molecule, is frequently expressed on infiltrating cells that often make close contact with breast carcinoma cells. To determine whether Sn could act as a specific receptor for ligands on breast cancer cell lines, binding assays were performed with a recombinant form of the protein fused to the Fc portion of human immunoglobulin G1 (IgG1) (Sn-Fc). Sn-Fc was found to bind specifically and in a sialic acid-dependent manner to the breast cancer cell lines MCF-7, T47.D and BT-20 both in solid- and solution-phase binding assays. To investigate the nature of the sialoglycoproteins recognized by Sn on breast cancer cells, MCF-7 cells were labelled with [6-3H]glucosamine. Following precipitation with Sn-Fc, a major band of approximately 240000 MW was revealed, which was shown in reprecipitation and Western blotting experiments to be the epithelial mucin, MUC1.

Blotting, Western↗

PubMed1998

Comparative analysis of integrins in vitro and in vivo in uveal and cutaneous melanomas.

Changes in integrin expression have been shown to be important for the growth and metastatic capacity of melanoma cells. In this study, we have examined the expression of alphav integrins by three uveal and four cutaneous malanoma lines. No lines expressed alphavbeta6 and only TXM13, a cutaneous line, expressed alphavbeta8. All lines expressed alphavbeta5 and alphavbeta3 (four out of four cutaneous, two out of three uveal) or avpl (OM431, an uveal line). Thus, OM431 is the second uveal melanoma we have described that expresses alphavbeta1 and this, we report again, functions as an alternative vitronectin/fibronectin receptor. Subcutaneous growth of cell lines in athymic mice correlated with an alphavbeta3-positive, alphavbeta1 -negative phenotype. Analysis of clinical material from cutaneous melanoma showed that although alphav expression was increased in 88% of metastases, this could not all be explained by up-regulation of alphavbeta3, with only 2 out of eight skin metastases expressing this heterodimer. Using antibody SZ.21, which as we report here works in archival material, only 1 out of 15 uveal metastases expressed detectable beta3. Thus, acquisition of alphavbeta3 expression, which has been implicated in cutaneous melanoma progression, may not be required for development of metastases from uveal melanoma or indeed for skin, as distinct from lymph node, metastases of cutaneous melanoma.

Animals↗

PubMed1995

Nitric oxide synthase activity in human breast cancer.

Nitric oxide (NO) is generated by a family of isoenzymes (NO synthases) expressed in a wide range of mammalian cells. We have recently reported NO synthase expression in human gynaecological cancers. In this study we have assessed the activity and distribution of NO synthase in a series of human breast tumours and in normal breast tissue. Calcium-dependent (constitutive) and -independent (inducible) NO synthase activity, as well as NO biosynthesis, was high in invasive tumours compared with benign or normal tissue. Furthermore, for invasive ductal carcinomas, NO biosynthesis was significantly greater for grade III compared with grade II tumours. Immunohistochemical investigations revealed immunolabelling with a monoclonal antibody to murine inducible NO synthase predominantly within tumour-associated macrophages. Immunolabelling with a polyclonal antiserum raised against rat brain NO synthase was also observed in vascular endothelial and myoepithelial cells. Thus NO synthase is expressed in human breast tumours, where its presence correlates with tumour grade.

Amino Acid Oxidoreductases↗

PubMed1992

Evaluation of PGP9.5 in the diagnosis of Hirschsprung's disease.

The ability of an acetylcholinesterase-stained frozen section to detect an increase in large cholinergic nerve fibres within the muscularis mucosae and extending into the lamina propria was a significant step forward in the diagnosis of Hirschsprung's disease (HD). However, such frozen section diagnosis is not always possible. The purpose of this study was to assess the ability of PGP9.5 to detect this pattern of mucosal nerve fibre staining immunohistochemically. Sixty-four specimens were included in the study. Twenty-six of these had been diagnosed as HD by conventional means. All cases were stained immunohistochemically with PGP9.5, S100, and anti-neurofilaments (NF). Twenty-four cases of HD were also stained with neurone-specific enolase (NSE). PGP9.5 reliably stained fibres in the mucosal and submucosal plexuses, and ganglion cells, when the latter were present. This positive staining of ganglion cells was more intense than that seen with NSE, and the positive fibre staining was more intense than that seen with NF. Increased lamina propria fibres were detected with PGP9.5 in only 37 per cent of HD cases compared with S100 positive staining in 60 per cent of cases. However, when S100 staining was assessed alone, it gave a higher false-negative rate in diagnosing HD than PGP9.5 used alone. Therefore we would recommend the use of PGP9.5 and S100 together for the immunohistochemical diagnosis of HD in formalin-fixed biopsies.

Adolescent↗

PubMed1992

An immunohistochemical investigation of diagnostic biopsy material taken from short and long term survivors with small cell lung cancer.

An immunohistochemical study has been carried out on fibre optic-biopsy specimens from patients with small cell lung cancer (SCLC) who had either died within 3 months, or who had survived more than 2 years. Long term survivors (LTS) were identified from completed clinical trials at major UK centres and were matched for age and sex within the trial with short term survivors (STS). The panel of immunohistochemical markers included those previously reported to be associated with prognosis, and reagents representative of both neuroendocrine and epithelial differentiation. A preliminary screen of 17 antibodies identified 11 as consistently reactive on paraffin-embedded material using streptavadin-biotin immunoperoxidase. Of 186 identified patients, 110 biopsy samples were retrieved. Of these, 70 gave sufficient material for analysis. All sections were scored by three observers without knowledge of the prognosis. The analysis failed to identify any antigen whose expression was correlated with prognosis. We conclude that, in fibre-optic biopsy specimens, immunohistochemical analysis does not add prognostic information in SCLC.

Antibodies, Monoclonal↗

PubMed1991

The differential diagnosis of epithelial-type mesothelioma from adenocarcinoma and reactive mesothelial proliferation.

Three monoclonal antibodies (mAbs) have been applied to 14 adenocarcinomas, eight mesotheliomas, and nine reactive mesothelial proliferations. One of these, AUA1, is a novel antibody in this context. It was found in 13/14 of the adenocarcinomas but not in benign or malignant mesothelial tissue, making it a potential substitute for carcinoembyronic antigen (CEA) in the differential diagnosis of mesotheliomas from adenocarcinomas. In addition, the lesions were stained using an argyrophil technique to identify nucleolar organizer regions. The results of counting these showed a small but significant overlap in their numbers between malignant and benign mesothelial proliferations. Although there was a difference overall in the numbers of nucleolar organizer regions in adenocarcinomas and mesotheliomas, on the one hand, and benign hyperplasias, on the other, this overlap considerably reduces their value in assessing individual difficult cases.

Adenocarcinoma↗

PubMed1991

Morphology and immunohistochemistry of carcinoma in situ adjacent to testicular germ cell tumours in adults and children: implications for histogenesis.

Observations differ on the pre-invasive malignant lesions associated with the various categories of testicular germ cell tumours. Such lesions have been found to be similar in appearance and are assumed to be composed of multipotent cells, or conversely a distinctive pre-invasive stage has been reported in association with each form of germ cell neoplasm. This study was undertaken to see whether distinctive morphological and immunohistochemical features of carcinoma in situ adjacent to various categories of germ cell tumours could be established. Carcinoma in situ adjacent to seminomas, teratomas and mixed germ cell tumours in 18 adults was indistinguishable morphologically. Placental alkaline phosphatase was demonstrated immunohistochemically but vimentin and low molecular weight cytokeratins were uniformly absent in these abnormal germ cells from all three groups. These findings support the concept of a multipotent pre-invasive malignant cell for both seminoma and teratoma in the adult. Carcinoma in situ was not seen adjacent to 15 spermatocytic seminomas, nor was placental alkaline phosphatase demonstrated in tubules adjacent to these tumours. These negative findings are additional evidence that spermatocytic seminoma differs from classical seminoma in its histogenesis. Carcinoma in situ, as defined morphologically and immunohistochemically in adults, was not identified adjacent to yolk sac tumours and differentiated teratomas in 20 prepubertal testes. The possibility that pre-invasive malignancy in children may not resemble that in adults must be considered when assessing the malignant potential of cryptorchid testes on biopsies taken during orchidopexy.

Adult↗

PubMed1991

Differential expression of antigens by human small cell lung carcinoma in sections of tumour and in serous effusions.

Antigen expression by human small cell lung carcinoma cells in serous effusions was determined by staining smears of cells with a panel of four monoclonal antibodies including UJ13A (cluster 1 antigen) and CAM 5.2 (anti-cytokeratin) using an immunoalkaline phosphatase technique. This was quantified by counting the proportion of stained and unstained tumour cells and was compared with that of small cell lung carcinoma in sections of solid tumour. Differential expression of antigen expression was noted with significantly fewer small cell carcinoma cells in serous effusions staining with UJ13A or CAM 5.2. The reasons for this differential expression are unknown, but may reflect adaptation to a different environment or be a prerequisite for spread to serous space.

Antigens, Neoplasm↗

PubMed1991

The expression of small cell lung cancer related antigens in foetal lung and kidney.

The presence and distribution of antigens recognised by the antibodies submitted to the Second International Workshop on Small Cell Lung Cancer Antigens has been mapped on human foetal lung and kidney using a streptavidin immunoperoxidase technique on frozen tissue sections. Thirteen of the antibodies showed no staining at all on these tissues and five showed staining of all structures in all tissues of equal intensity. The staining patterns of the remaining antibodies could be divided into several groups. All the cluster 1 antibodies recognising the neural cell adhesion molecule (NCAM) gave a distinctive staining pattern in both tissues. The cluster 2 antibodies and several groups of unclustered antibodies gave varying pure epithelial patterns of staining. The cluster w4 and 5 antibodies gave an interesting staining pattern which included some of the distinctive features of the cluster 1 pattern plus some epithelial expression. Various less distinctive patterns were also observed. Serial sections of foetal lung which had been stained with NCC-LU-246 (cluster 1), anti-desmin, and anti-neurofilament antibodies were also studied to attempt to determine the nature of the cells expressing NCAM in the foetal lung.

Antibodies, Monoclonal↗

PubMed1990

Human thymocyte development in mouse organ cultures.

A novel system to study human thymocyte development is described in which embryonic mouse thymic rudiments are seeded with human precursor cells in vitro. In these cultures human thymocytes proliferate extensively (greater than 20-fold increase in cell number) and mature, as evidenced by the accumulation of double and single positive (CD4+ and/or CD8+) cells. Data presented here suggest that the survival and ordered development of the mature human thymocytes in chimeric thymuses is dependent on human stromal elements. Immature CD4-CD8- human thymocytes failed to colonize or minimally recolonized mouse thymic lobes unless provided with high density (greater than 1.077 g/ml) human thymic cell fractions. These fractions contain multicellular complexes of epithelial/nurse cells, thymocytes, and dendritic cells/macrophages which dramatically enhanced the recolonizing capacity of purified CD4-CD8- thymocytes. The chimeric organ culture system described here provides not only a new approach for studying human T cell ontogeny but also a direct means for the future dissection of stromal interactions necessary for successful transition of precursor cells (CD4-CD8-) to immature double positive (CD4+CD8+) and mature single positive cells (CD4+ or CD8+) in the thymus.

Animals↗

PubMedDate not supplied

Expression of the hypervariable PUM locus in normal and malignant lung: the tumor-associated epitopes are present but masked in normal tissue.

A single highly polymorphic gene locus PUM codes for a family of mucin-type glycoproteins present in human urine. These glycoproteins can be detected after electrophoresis using a group of monoclonal antibodies which show marked tumour specificity on immunohistology and include the HMFG and Ca antibodies (Swallow et al., 1986, 1987). Here we show by electrophoretic analysis of lung specimens and urine samples from nine individuals, that the PUM locus is expressed both in malignant and in normal lung. In contrast immunohistology of frozen sections of normal lung showed very little staining using the same antibodies, occasional reactive type 2 pneumocytes alone staining, whilst the carcinoma material showed strong staining in each case. However, after formalin fixation much more staining was observed in normal lung, all type 1 and 2 pneumocytes being stained. These observations suggest a difference in accessibility of the epitopes in normal and malignant lung, rather than a difference in expression of the PUM gene.

Antigens, Neoplasm↗