Generation and initial characterization of conditionally immortalized chromaffin cells

Abstract

Adrenal chromaffin cells have been successfully used to attenuate chronic pain when transplanted near the spinal cord, but primary cells are neither homogeneous nor practical for routine use in human therapy. Conditional immortalization with the temperature-sensitive allele of the large T antigen (tsTag) and creation of stable chromaffin cell lines would advance our understanding of both the use and limits of cell lines that contain this immortalization gene for such therapies. Cultures of embryonic day 17 rat adrenal and neonatal bovine adrenal cells were immortalized with the temperature-sensitive allele of SV40 tsTag and chromaffin cell lines established. The rat chromaffin line, RAD5.2, and the bovine chromaffin cell line, BADA.20, both expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines, dopa-beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). At permissive temperature (33 degrees C), these chromaffin cells are proliferative, have a typical rounded chromaffinlike morphology, and contain detectable TH-, DbetaH-, and PNMT-ir. At nonpermissive temperature (39 degrees C), these cells stop proliferating, decrease Tag expression, and change the expression of TH-, DbetaH-, and PNMT-ir in vitro, suggesting increased differentiation at nonpermissive temperature. The chromaffin cell lines also express immunoreactivity for the opioid met-enkephalin (ENK) at permissive and nonpermissive temperatures. The expression of TH-ir in the bovine chromaffin cells is upregulated by the addition of dexamethasone (DEX) or forskolin during differentiation; TH-ir is not affected by the addition of DEX or forskolin in the rat chromaffin cells. The addition of forskolin during differentiation upregulates the expression of DbetaH-ir in the rat chromaffin cells. PNMT-ir is not affected by differentiation or agents in either cell line. However, catecholamine synthesis was not detectable by high-performance liquid chromatography, suggesting incomplete differentiation under current conditions, or influence by continued low levels of Tag expression. Both cell lines have been carried over many passages in vitro for more than 3 years and were repeatedly frozen and thawed. These data describe an initial step in the conditional immortalization of chromaffin cells that can maintain the phenotype of primary chromaffin cells in vitro over long periods. The use of such chromaffin cell lines that are able to deliver neuroactive molecules offers a novel approach to pain management.

Induction of Tyrosine Hydroxylase Gene Expression by Glucocorticoids in the Perinatal Rat Brain is Age-Dependent

“…The reasons for these contradictions as well as T for the discrepancies between in vitro and in vivo studies are not clear. It is possible that cultured cells acquire sensitivity to the hormone along their transformation into immortalized state, during which some degree of cell dedifferentiation occurs [28]. Besides, cultured cells lack intercellular regulatory contacts that are normal for adult brain cells that may limit the activation of TH by glucocorticoids.…”

Cell therapy for neuropathic pain in spinal cord injuries

“…A similar approach was used to immortalise embryonic rat and bovine chromaffin cells [66], using SV40tsTag for immortalisation. The cell biology and developmental responsiveness of primary chromaffin cells during differentiation [67] reveals clues to the differentiation programme of conditionally immortalised chromaffin cell lines in vitro.…”

“…Although originally reported to lack phenylethanolamine N-methyltransferase (PNMT) and Bovine [85] Human [86] Porcine [87] Midbrain [88] Formalin [18] Nerve injury [17] SCI [31][32][33] Cancer [35,89,90] Arthritis [24,38,91] Met-enkephalin [19] Catecholamines [20] GABA? [26] Enkephalinase [92] c-fos [ Tail-flick or chemical induction [44,[46][47][48] Partial nerve injury (CCI) [45] Formalin [48] Catecholamines [44,45] Opioids [46] Conditionally immortalised Embryonic rat raphe, SV40tsTag [93,94] Partial nerve injury (CCI) [ Embryonic rat and bovine chromaffin [66] Partial nerve injury (CCI) [72] ?? ?…”

“…The enzyme tyrosine hydroxylase (TH; EC1.14.3.x) catalyses the rate-limiting step [68] in the biosynthesis of catecholamines in chromaffin cells in the adrenal medulla [69,70], and has been used as one of the antigenic markers for the mature chromaffin phenotype of primary rat and bovine chromaffin cells in vitro [71], as well as the dopamine β-hydroxylase (DβH) and PNMT enzymes. However, even with upregulation of enzyme expression that accompanies differentiation, these conditionally immortalised chromaffin rat and bovine cells do not synthesise catecholamines under in vitro conditions [66]. These conditionally immortalised cells express very low levels of TH.…”

Past, Present and Future of Human Chromaffin Cells: Role in Physiology and Therapeutics

“…Since the limited amount of tissue is one of the main limitations of the strategies mentioned above, efforts to create a cell line of sympathetic neurons have led to the production of immortalized chromaffin cells (Eaton et al 2000). However, the use of these cells has the shortcoming that they may induce tumors in the CNS, and several measures have been proposed to limit the expandability of these cells (Eaton and Duplan 2004).…”