Guidelines for the use of cell lines in biomedical research.

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

CROC-4: a novel brain specific transcriptional activator of c-fos expressed from proliferation through to maturation of multiple neuronal cell types.

A novel, brain-specific cDNA, denoted CROC-4, was cloned from human brain by a contingent replication of cDNA procedure capable of detecting transcriptional activators of the human c-fos proto-oncogene promoter. CROC-4 encoded an 18-kDa serine/threonine-rich polypeptide containing a P-loop motif and an SH3-binding region with phosphorylation sites for a variety of protein kinases (cdc2, CDK2, MAPK, CDK5, protein kinase C, Ca(2+)/calmodulin protein kinase 2, casein kinase 2) involved in cell proliferation and differentiation. Immunohistochemistry revealed that during early development, expression was associated with proliferating and migrating cells throughout the rodent brain, initially appearing in the proliferative ventricular zones. During late development and in adult human brain, CROC-4 was expressed in diverse brain regions including the thalamus, subthalamic nucleus, corpus callosum, substantia nigra, caudate nucleus, amygdala, and hippocampus. The association of CROC-4 expression with proliferating regions of developing brain and retention in regions of the adult brain, as well as the punctate nuclear location, suggest that CROC-4 participates in brain-specific c-fos signaling pathways involved in cellular remodeling of brain architecture.

Expression of doublecortin correlates with neuronal migration and pattern formation in diverse regions of the developing chick brain.

The development of functional layers in the brain involves spatially and temporally regulated gene expression. Through cDNA library screening, we have identified genes that are expressed in a neural-specific manner during brain development. Sequencing and expression data indicate that one of the clones, 18C15, is the chick homologue of doublecortin, a human X-linked gene found to be mutated in subcortical laminar heterotopia (double cortex syndrome) and lissencephaly. The 18C15 mRNA contains multiple motifs that are known to regulate mRNA stability in response to inductive signals, and these motifs are conserved between the chick and human sequences. Doublecortin is found to be expressed at peak levels during early development of the cerebellum and forebrain, and is expressed in other regions including the tectum, spinal cord, and dorsal root ganglia. This study demonstrates both spatial and temporal regulation of doublecortin expression in the chick, which is associated with early events in brain development, including neuronal migration.

Glucocorticoids differentially inhibit expression of the RET proto-oncogene.

The RET proto-oncogene encodes a receptor tyrosine kinase activated by the binding of factors from the glial cell line-derived neurotrophic factor (GDNF) family to receptor-alpha components such as GDNF family receptor alpha-1 (GFR alpha-1). Mutations within the sequence of the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (MEN 2), an inherited tumor syndrome characterized by the development of medullary thyroid carcinoma (MTC) and other neuroendocrine tumors. Despite Northern analysis showing that RET is expressed in the majority of MTCs, the factors regulating this expression are poorly understood. To address this issue we examined RET expression in response to glucocorticoids in the TT cell line, derived from a metastatic MTC. The synthetic glucocorticoid dexamethasone was found to reduce RET expression at both mRNA and protein levels. This effect was dose responsive and maximal at 24 h. The reduction in RET mRNA was shown to be specific to glucocorticoids and was also seen in a primary MTC culture. Nuclear run-on studies revealed the reduction in steady-state RNA to be due to a decrease in RET mRNA transcription and the effect was shown to be independent of new protein synthesis or RNA stability. Dexamethasone was also found to exert an inhibitory effect upon cell growth, suggesting a potential use for glucocorticoids in the treatment of medullary carcinoma and MEN 2.