Isolation and Growth of Smooth Muscle-Like Cells Derived from Tuberous Sclerosis Complex-2 Human Renal Angiomyolipoma

Abstract

Tuberous sclerosis complex (TSC) is a tumor suppressor gene disorder characterized by mutations in the TSC1 or TSC2 genes. These mutations lead to the development of benign tumors involving smooth muscle cells, causing life-threatening lymphangioleiomyomatosis. We isolated and characterized two types of cells bearing a mutation in TSC2 exon 18 from a renal angiomyolipoma of a TSC patient: one population of alpha-actin-positive smooth muscle-like cells with loss of heterozygosity for the TSC2 gene (A(+) cells) and another of nonloss of heterozygosity keratin 8/18-positive epithelial-like cells (R(+) cells). Unlike control aortic vascular smooth muscle cells, A(+) cells required epidermal growth factor (EGF) to grow and substituting EGF with insulin-like growth factor (IGF)-1 failed to increase the cell number; however, omission of EGF did not cause cell loss. The A(+) cells constantly released IGF-1 into the culture medium and constitutively showed a high degree of S6K phosphorylation even when grown in serum-free medium. Exposure to antibodies against EGF and IGF-1 receptors caused a rapid loss of A(+) cells: 50% by 5 days and 100% by 12 days. Signal transduction mediated by EGF and IGF-I receptors is therefore involved in A(+) cell survival. These results may offer a novel therapeutic perspective for the treatment of TSC complications and lymphangioleiomyomatosis.

Survivin Expression in Tuberous Sclerosis Complex Cells

“…However, its expression increases dramatically when the cells are exposed to agents that affect their function, such as LY, PD, and the mTOR inhibitor rapamycin. At a dose of 5 ng/mL, rapamycin affects the proliferation of smooth muscle TSC2 -/-A + cells (46), but LY and PD do not; the addition of anti-EGFR to the growth medium affects the rate of proliferation and promotes the expression of survivin. Both rapamycin and anti-EGFR increase survivin levels without affecting the constitutively expressed Smac/DIABLO, but their effects are time related: after five days' treatment, the survivin-promoting effects of anti-EGFR or rapamycin have almost vanished, whereas Smac/DIABLO is still ex- the cells washed once, and then fresh medium was added and collected 24 h later for IGF-1 assay.…”

“…Two pure homogeneous cell populations were isolated from the angiomyolipoma of a TSC2 patient and characterized by sub-cloning at limiting dilution (46). One population consisted of elongated, flat cells highly positive for smooth muscle-specific ␤-actin ( Figure 2a); the cells also are labeled by anti-HMB45 antibody (46), a TSC marker (49,50), but are negative for S100 and keratin 8/18.…”

“…One population consisted of elongated, flat cells highly positive for smooth muscle-specific ␤-actin ( Figure 2a); the cells also are labeled by anti-HMB45 antibody (46), a TSC marker (49,50), but are negative for S100 and keratin 8/18. These smooth muscle-like cells (A + ) are LOH (a mutation on chromosome 16p13.3 at exon 18 consisting of an A/T substitution) and do not express tuberin (46). Conversely, the epithelioid cells (R + ) were highly positive for keratin 8/18 and RhoA (Figure 2b), and negative for ␤-actin and S100; they are not LOH and express tuberin in both its phosphorylated and nonphosphorylated forms (46).…”

“…Adding EGF to the culture medium allows the proliferation of A + cells (46) and, as described above, keeps the expression of survivin at undetectable levels. However, when these cells were exposed for 48 h to EGFR antibodies (5 g/mL) raised against the receptor domain that interacts with the ligand (clone 225), survivin expression was greatly enhanced ( Figure 6A).…”

“…However, this feedback regulation is overcome if TSC2 -/-A + cells are exposed to antibodies to IGF-1R, which increases the released amount by more than 400%. We have reported previously that TSC2 -/-A + cells exposed to anti-IGF-1R gradually die in 12-14 days (46), and so the autocrine release of IGF-1 seems to be a powerful survival mechanism activated by TSC2 -/-smooth muscle cells which are tightly packed in the thick-walled vessels of TSC angiomyolipoma and lymphangioleiomyomatosis (LAM) nodules. Survivin originally was isolated as a member of IAP family, and it was suggested that it plays a fundamental role in inhibiting apoptosis and regulating the mitotic process and cell proliferation (19,20).…”