Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

Differential subtraction display: a unified approach for isolation of cDNAs from differentially expressed genes.

We have developed a novel efficient approach, termed differential subtraction display, for the identification of differentially expressed genes. Several critical parameters for the reproducibility and enhanced sensitivity of display, as well as steps to reduce the number of false positive cDNA species, have been defined. These include- (a) use of standardized oligo(dT)-primed cDNA pools rather than total RNA as the starting material for differential display, (b) critical role of optimal cDNA input for each distinct class of primers, (c) phenomena of primer dominance and interference, and (d) design of a novel set of enhanced specificity anchor primers. Introduction of an efficient subtractive hybridization step prior to cloning of cDNA species enriches the bona fide cDNA species that are either exclusively present in one sample (+/-) or show altered expression (up-/down-regulation) in RNA samples from two different tissues or cell types. This approach, in comparison to differential display, has several advantages in terms of reproducibility and enhanced sensitivity of display coupled to the cloning of enriched bona fide cDNA species corresponding to differentially expressed RNAs.

Hair follicle growth controls.

Research in hair biology has embarked in the pursuit for molecules that control hair growth. Many molecules already have been associated with the controls of hair patterning, hair maturation, and hair cycling and differentiation. Knowing how these molecules work gives us the tools for understanding and treating patients with hair disorders.

Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts.

Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

SV40-mediated immortalization of human fibroblasts.

We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

Altered chromosome 6 in immortal human fibroblasts.

Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.