Molecular indexing of human genomic DNA.

Molecular indexing sorts DNA fragments into subsets for inter-sample comparisons. Type IIS or interrupted palindrome restriction endonucleases, which result in single-stranded ends not including the original recognition sequence of the enzyme, are used to produce the fragments. The ends can then be any sequence but will always be specific for a given fragment. Fragments with particular ends are selected by ligation to a corresponding indexing adapter. We describe iterative indexing, a new process that after an initial round of indexing uses a Type IIS restriction endonuclease to expose additional sequence for further indexing. New plasmids, pINDnn, were produced for novel use as indexing adapters. Together, the plasmids index all 16 possible dinucleotides. Their large size can be increased by dimerisation in vitro and allows the isolation of indexed material by size separation. Fragments produced from human genomic DNA by Type II restriction endonucleases were sorted using six bases in total to a possible enrichment of 1920-fold. By comparison with the public human sequence databases, fidelity of indexing was shown to be high and was tolerant of repetitive sequences. Genome-wide comparisons on a candidate or non-candidate basis are made possible by this approach.

Induction of the metastatic phenotype by transfection of a benign rat mammary epithelial cell line with the gene for p9Ka, a rat calcium-binding protein, but not with the oncogene EJ-ras-1.

The rat mammary epithelial cell line, Rama 37, yields benign, non-metastasizing adenomatous tumours in syngeneic Wistar-Furth rats. Transfection of this line with a drug resistance plasmid containing both the gene for resistance to Geneticin (neo) and the gene for p9Ka (pSV2neo-p9Ka), a rat calcium-binding protein, or with a similar plasmid containing neo and the oncogene EJ-ras-1 (pSV2neo-ras) yields drug-resistant transformants that express high levels of the p9Ka or EJ-ras-1 mRNAs and proteins. These transfected cells all produce tumours when injected at subcutaneous sites with a shorter median latent period than the tumours produced by the parental untransfected Rama 37 cells in syngeneic hosts. Cells transfected with pSV2neo-p9Ka yield a higher incidence of tumours than untransfected Rama 37 cells, many of which metastasize to lungs and/or lymph nodes in syngeneic rats. However, cells transfected with pSV2-neo-ras or pSV2neo plasmid alone yield tumours that fail to metastasize. Immunofluorescent studies suggest an association of p9Ka with the cytoskeleton, as depicted by F-actin staining with the reagent phalloidin. It is suggested that the transfection of copies of the gene for the rat calcium-binding protein p9Ka can enhance the tumorigenic potential and induce the metastatic phenotype in this rat mammary model, whereas transfection of control plasmid DNA or the oncogene EJ-ras-1 fails to induce the metastatic phenotype, although EJ-ras-1 transfectants, like those containing p9Ka, possess increased growth properties in vivo.

Immunocytochemical distribution of the calcium-binding protein p9Ka in normal rat tissues: variation in the cellular location in different tissues.

The family of S-100-related proteins consists of a number of small potential calcium-binding proteins of unknown function. Elevated expression of one of these proteins, p9Ka, or of its mRNA, correlates with the metastatic potential of cultured mammary epithelial cells from rat or mouse. Over-expression of p9Ka by transfection of benign rat mammary epithelial tumor cells with the gene for p9Ka induces the metastatic phenotype. At present there is little information on the occurrence of p9Ka in normal rat tissues. A specific antiserum immunocytochemically detects p9Ka intracellularly in most normal adult rat tissues studied, including smooth muscle, brown adipose tissue, and liver. In other tissues, p9Ka is localized specifically to some absorptive and keratinized epithelia, the acid-secreting parietal cells of the stomach, the neuronal cells within plexuses of the autonomic nervous system, and a proportion of cells of the immune system in spleen, lymph nodes, bone marrow, and blood. p9Ka is found widely in both arteries and veins, particularly in the smooth muscle and in the endothelium of smaller veins. In mammary gland, the pattern of staining suggests that p9Ka is extracellularly located in a region surrounding the ducts.

Interactions in vitro of p9Ka, the rat S-100-related, metastasis-inducing, calcium-binding protein.

The S-100 proteins are a structurally related family displaying diverse intracellular and extracellular interactions. One such protein p9Ka (also known as calvasculin), or its mRNA (also known as CAPL, 42A, 18A2, mts, pEL 98), becomes elevated upon changes in the growth and differentiation of cells. Overexpression of p9Ka in benign rat mammary cells induces the metastatic phenotype. In order to help understand the role of p9Ka in these processes, the molecular properties of recombinant rat p9Ka have been studied. Recombinant p9Ka forms multimers in vitro, which are not due to intermolecular disulfide bridges, it binds 2 mol of calcium ions/mol of protein, and the binding of calcium ions is strongly antagonized by monovalent and divalent cations tested. Immunofluorescence studies indicate that p9Ka is located on cytoskeletal elements in a pattern which is identical to actin filaments stained with phalloidin. In vitro, it is shown that recombinant p9Ka binds to sites on at least two intracellular polypeptides. These sites display the same binding capacity for p9Ka in extracts of cultured rat mammary cells which show widely differing levels of expression of natural p9Ka. The results suggest that the production of p9Ka, and not of its target molecules, may be associated with the changes seen in cultured cells.

Mutation in the PTEN/MMAC1 gene in archival low grade and high grade gliomas.

The PTEN gene, located on 10q23.3, has recently been described as a candidate tumour suppressor gene that may be important in the development of advanced cancers, including gliomas. We have investigated mutation in the PTEN gene by direct sequence analysis of PCR products amplified from samples microdissected from 19 low grade (WHO Grade I and II) and 27 high grade (WHO grade III and IV) archival, formalin-fixed, paraffin-embedded gliomas. Eleven genetic variants in ten tumours have been identified. Eight of these are DNA sequence changes that could affect the encoded protein and were present in 0/2 pilocytic astrocytomas, 0/2 oligoastrocytomas, 0/1 oligodendroglioma, 0/14 astrocytomas, 3/13 (23%) anaplastic astrocytomas and 5/14 (36%) glioblastomas. PTEN mutations were found exclusively in high grade gliomas; this finding was statistically significant. Only two of the PTEN genetic variants have been reported in other studies; two of the genetic changes are in codons in which mutations have not been found previously. The results of this study indicate that mutation in the PTEN gene is present only in histologically more aggressive gliomas, may be associated with the transition from low histological grade to anaplasia, but is absent from the majority of high grade gliomas.

Characterisation of molecular alterations in microdissected archival gliomas.

Classification of gliomas according to their molecular characteristics may be important in future histopathological diagnosis. However, gliomas frequently display heterogeneity at the histological, biological and molecular level. In this study of archival diagnostic gliomas, precision microdissection was used to enrich samples in the most malignant cells or to investigate intratumoural histological heterogeneity. Analysis of tumour samples microdissected from the most aggressive regions, representative of the histopathological diagnosis, revealed PTEN mutations in 4/14 anaplastic astrocytomas, 4/13 glioblastomas and 1 gliosarcoma, but not in 19 low-grade gliomas. Using a novel PCR procedure and direct sequence analysis of the entire coding sequence, TP53 mutations were detected in 1/3 pilocytic astrocytomas, 3/13 astrocytomas, 4/14 anaplastic astrocytomas, 5/13 glioblastomas and 1 gliosarcoma. All but one of the tumours with TP53 mutation showed p53 immunopositivity, but 5 low-grade and 10 high-grade gliomas had p53 protein nuclear accumulation in the absence of detectable mutation. p53 status was unrelated to p21 expression. Neither PTEN nor TP53 mutations influenced the proliferative index or microvessel density of high-grade astrocytomas. Unusual findings include: TP53 mutation in a juvenile pilocytic astrocytoma; TP53 and PTEN mutations in a de novo glioblastoma, a gliosarcoma with identical mutations in gliomatous and sarcomatous components, and an infratentorial anaplastic astrocytoma with an earlier supratentorial grade II astrocytoma bearing the same TP53 mutation but not the PTEN mutation or loss of heterozygosity (LOH) of 10q23. Similarly, the transition to high-grade histology was associated with acquisition of PTEN mutations and 10q23.3 LOH in two de novo high-grade tumours with regions of low-grade histology.