Derek McCormick

Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis.

Papillon-Lefèvre syndrome, or keratosis palmoplantaris with periodontopathia (PLS, MIM 245000), is an autosomal recessive disorder that is mainly ascertained by dentists because of the severe periodontitis that afflicts patients. Both the deciduous and permanent dentitions are affected, resulting in premature tooth loss. Palmoplantar keratosis, varying from mild psoriasiform scaly skin to overt hyperkeratosis, typically develops within the first three years of life. Keratosis also affects other sites such as elbows and knees. Most PLS patients display both periodontitis and hyperkeratosis. Some patients have only palmoplantar keratosis or periodontitis, and in rare individuals the periodontitis is mild and of late onset. The PLS locus has been mapped to chromosome 11q14–q21 (refs 7, 8, 9). Using homozygosity mapping in eight small consanguineous families, we have narrowed the candidate region to a 1.2-cM interval between D11S4082 and D11S931. The gene (CTSC) encoding the lysosomal protease cathepsin C (or dipeptidyl aminopeptidase I) lies within this interval. We defined the genomic structure of CTSC and found mutations in all eight families. In two of these families we used a functional assay to demonstrate an almost total loss of cathepsin C activity in PLS patients and reduced activity in obligate carriers.

The clinical significance of cathepsin S expression in human astrocytomas.

Early local invasion by astrocytoma cells results in tumor recurrence even after apparent total surgical resection, leading to the poor prognosis associated with malignant astrocytomas. Proteolytic enzymes have been implicated in facilitating tumor cell invasion and the current study was designed to characterize the expression of the cysteine proteinase cathepsin S (CatS) in astrocytomas and examine its potential role in invasion. Immunohistochemical analysis of biopsies demonstrated that CatS was expressed in astrocytoma cells but absent from normal astrocytes, oligodendrocytes, neurones and endothelial cells. Microglial cells and macrophages were also positive. Assays of specific activity in 59 astrocytoma biopsies confirmed CatS expression and in addition demonstrated that the highest levels of activity were expressed in grade IV tumors. CatS activity was also present in astrocytoma cells in vitro and the extracellular levels of activity were highest in cultures derived from grade IV tumors. In vitro invasion assays were carried out using the U251MG cell line and the invasion rate was reduced by up to 61% in the presence of the selective CatS inhibitor 4-Morpholineurea-Leu-HomoPhe-vinylsulphone. We conclude that CatS expression is up-regulated in astrocytoma cells and provide evidence for a potential role for CatS in invasion.

GLIOMA INVASION IN VITRO IS MEDIATED BY CD44–HYALURONAN INTERACTIONS

Invasion is a clinically important problem contributing to mortality and morbidity in patients with gliomas, but the mechanism(s) by which glioma cells invade surrounding brain structures is poorly understood. Various experimental models have been used in attempts to elucidate the process of glioma invasion. An in vitro model which is increasingly being employed involves measurement of the rate of invasion of tumour cells through Matrigel®—a complex mixture of extracellular matrix components derived from the Engelbroth–Holm–Swarm (EHS) sarcoma. This model has been used to examine the possibility that extracellular hyaluronan (HA) might facilitate the invasive behaviour of human glioma cells. The major component of Matrigel® is laminin, with smaller amounts of collagen IV, heparan sulphate proteoglycans, entactin, and nidogen, but it lacks HA. In our experiments, we have incorporated HA into Matrigel® and have measured its effect on the rate of invasion of human glioma cells in a modified Boyden chamber assay system. The incorporation of HA (50–800 mg/cm2) resulted in a dose‐dependent increase in invasion. Invasion was enhanced by up to 70 per cent in comparison with HA‐free Matrigel®. Since CD44 is a major HA receptor expressed on gliomas, it might have a role in the HA‐mediated facilitation of invasion. This was tested by blocking CD44 with specific antibody, which resulted in a 43 per cent reduction in invasion rate. We conclude that in an in vitro model system, HA enhances invasion of glioma cells and that the mechanism involves a CD44–HA interaction.

Cathepsin S expression: An independent prognostic factor in glioblastoma tumours - A pilot Study

Cysteine proteinases have been implicated in astrocytoma invasion. We recently demonstrated that cathepsin S (CatS) expression is up‐regulated in astrocytomas and provided evidence for a potential role in astrocytoma invasion (Flannery et al., Am J Path 2003;163(1):175–82). We aimed to evaluate the significance of CatS in human astrocytoma progression and as a prognostic marker. Frozen tissue homogenates from 71 patients with astrocytomas and 3 normal brain specimens were subjected to ELISA analyses. Immunohistochemical analysis of CatS expression was performed on 126 paraffin‐embedded tumour samples. Fifty‐one astrocytoma cases were suitable for both frozen tissue and paraffin tissue analysis. ELISA revealed minimal expression of CatS in normal brain homogenates. CatS expression was increased in grade IV tumours whereas astrocytoma grades I–III exhibited lower values. Immunohistochemical analysis revealed a similar pattern of expression. Moreover, high‐CatS immunohistochemical scores in glioblastomas were associated with significantly shorter survival (10 vs. 5 months, p = 0.014). With forced inclusion of patient age, radiation dose and Karnofsky score in the Cox multivariate model, CatS score was found to be an independent predictor of survival. CatS expression in astrocytomas is associated with tumour progression and poor outcome in glioblastomas. CatS may serve as a useful prognostic indicator and potential target for anti‐invasive therapy.

CD44 plays a role in adhesive interactions between glioma cells and extracellular matrix components

Glioma invasion is a complex process involving interactions of tumour cells with host cells and extracellular matrix (ECM). The initial event in the process is recognition and attachment of glioma cells to specific ECM molecules prior to migration into proteolytically modified matrix. In comparison with other tissues, brain ECM is a relatively amorphous matrix which contains glycosaminoglycans including hyaluronan (HA). Recently CD44 which is a transmembrane adhesion molecule found on a wide variety of cells, has been suggested as the principal cell surface receptor for HA. In the present in vitro investigation we have analysed the role of CD44 in adhesive interactions between human gliomas and ECM. Our experimental procedures included immunocytochemistry, immunoblotting, in vitro adhesion assay and flow cytometry. CD44 was expressed on the surface of all gliomas analysed (9) and the level of expression showed no correlation with tumour grade. Eighty, 95 and 120 kDa isoforms were demonstrated by immunoblotting. In an adhesion blocking assay it was found that ligation of CD44 with specific antibody resulted in reduced adhesion to hyaluronan. chondroitin sulphate, fibronectin, laminin, collagen IV and Matrigel™. We conclude that CD44 is involved in adhesion of glioma cells to a wide range of ECM components.

Secretion of cathepsin B by human gliomas in vitro

There is evidence from investigations of non‐CNS neoplasms that secreted proteolytic enzymes may facilitate tumour invasion by partially degrading extracellular matrix (ECM). Among the enzymes which may be involved are members of the cysteine proteinase superfamily and especially cathepsin B (CB). In the present investigation we have studied CB in human gliomas in vitro, concentrating particularly on CB secretion, as extracellular enzyme is of prime importance in this context. We have found that CB is secreted by gliomas in vitro as a latent zymogen, requiring activation. This has been confirmed by gel chromatography which indicated that CB is secreted as a 42 kDa proenzyme which may be proteolytically processed to an enzymatically active 29 kDa molecule. The inactive, high molecular weight, latent CB is stable at extracellular pH in contrast to the activated low molecular weight form which rapidly loses activity at this pH. We have also measured secretion of cysteine proteinase inhibitors (CPI), as their presence would have a direct influence on the effective activity of CB, and found that all of the gliomas secreted significant amounts of a CPI as assessed by papain inhibition. Our experiments suggest that a number of factors are involved in the regulation of extracellular glioma‐derived CB activity. These include: rate of secretion of pro‐CB, rate of CB activation, destabilization of CB at neutral pH and the presence of cysteine proteinase inhibitors.

Epidermal growth factor up-regulates CD44-dependent astrocytoma invasion in vitro

CD44/hyaluronan interactions and epidermal growth factor (EGF) stimulation are both known to enhance tumour invasion in vitro. The frequent amplification of the EGF receptor (EGFR) in high‐grade astrocytomas led to the examination of the hypothesis that CD44‐dependent astrocytoma invasion is regulated by EGF. It has been shown that human astrocytoma cells express only the standard (haemopoietic) form of CD44 (CD44s) and that EGF up‐regulates CD44 mRNA and protein in a time‐ and dose‐dependent (10–100 ng/ml) manner. EGF stimulation did not result in induction of additional splice variants. No EGF‐induced increase in CD44s was observed after treatment of cells with the wild‐type EGFR tyrosine kinase inhibitor Tyrphostin AG1478 (30 nM). Up‐regulation of CD44 by EGF is also prevented by the transcriptional inhibitor actinomycin D (5 µg/ml) and by blocking the MAP kinase (MAPK) pathway using the MEK inibitor U0126 (100 µM). CD44 up‐regulation was associated with a 50% increase in invasion through hyaluronan‐supplemented Matrigel™, which was abrogated by ligating CD44 with the specific antibody KM201. These results suggest that increased CD44 expression in response to EGF stimulation plays a significant role in astrocytoma invasion. Copyright

Use of recombinant granulocyte-colony stimulating factor to treat neutropenia occurring during craniospinal irradiation

PURPOSE To investigate the effectiveness of recombinant human Granulocyte-Colony Stimulating Factor, a hematopoietic growth factor which stimulates neutrophil production, in the treatment of neutropenia caused by Craniospinal Irradiation. METHODS AND MATERIALS Four consecutive patients who developed neutropenia (neutrophils less than 1.5 X 10(9)/l in peripheral blood) during craniospinal irradiation for primary intracranial tumors received intermittent subcutaneous injections of Granulocyte-Colony Stimulating Factor. Two of the patients had medulloblastoma, one had a primitive neuroectodermal tumor and the other a pinealocytoma. No patient received prior or concurrent chemotherapy. RESULTS In all cases peripheral blood neutrophil counts returned rapidly to normal levels following Granulocyte-Colony Stimulating Factor injections and treatment delays were therefore avoided. Platelet counts were unaffected by Granulocyte Colony Stimulating Factor treatment. In one case, slight elevation of peripheral blood monocyte and lymphocyte counts occurred after each Granulocyte-Colony Stimulating Factor injection. No toxicity was encountered. CONCLUSION Our results suggest that Granulocyte-Colony Stimulating Factor is a safe and effective treatment for neutropenia caused by extended field radiotherapy.

REDUCED MUSCLE α-GLUCOSIDASE (ACID-MALTASE) ACTIVITY IN HYPOTHYROID MYOPATHY

Abstract A detailed biochemical study of muscle from a patient complaining of disabling cramps revealed a lowered activity of α-glucosidase (acid-maltase), which was the only abnormality detected. A year after the onset of symptoms hypothyroidism was diagnosed. Treatment with L-thyroxine relieved all symptoms, and the muscle acid-maltase activity was found to be normal six months after the initiation of treatment.

A study of glutamine synthetase in normal human brain and intracranial tumours

A study of glutamine synthetase in normal human brain and intracranial tumours. Glutamine synthetase (GS) activity was measured in selected areas of three normal brains and in 262 biopsies from patients with suspected intracranial tumours. In general, levels were higher in grey matter than in white matter and the highest activities of all were found in the hypothalamus which is consistent with its high glutamatergic activity. In the biopsy material, GS activity was greatest in gliotic brain, in keeping with the predominantly astrocytic localization of the enzyme. High levels were also found in astrocytomas and oligodendrogliomas but there was considerable variation between tumours, suggesting a random loss of GS expression during neoplastic transformation or heterogeneity in their cellular origin. The immunocytochemical demonstration of GS in neoplastic oligodendrocytes and in meningioma cells argues against absolute cell‐type specificity for this enzyme.