We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-␣ 1 and -2 (Col1a1 and -2). Transforming growth factor ␤1 (TGF-␤), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-␤ increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, ␦EF1. TGF-␤ treatment or short hairpin RNAs targeting ␦EF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-␤ also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to ␦EF1. Interestingly, we noted that SIP1 is a target of microRNA-192 (miR-192), a key miR highly expressed in the kidney. miR-192 levels also were increased by TGF-␤ in MMC. TGF-␤ treatment or transfection with miR-192 decreased endogenous SIP1 expression as well as reporter activity of a SIP1 3 UTR-containing luciferase construct in MMC. Conversely, a miR-192 inhibitor enhanced the luciferase activity, confirming SIP1 to be a miR-192 target. Furthermore, miR-192 synergized with ␦EF1 short hairpin RNAs to increase Col1a2 E-box-luc activity. Importantly, the in vivo relevance was noted by the observation that miR-192 levels were enhanced significantly in glomeruli isolated from streptozotocin-injected diabetic mice as well as diabetic db/db mice relative to corresponding nondiabetic controls, in parallel with increased TGF-␤ and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-␤-induced Col1a2 expression by down-regulating E-box repressors.diabetic nephropathy ͉ mesangial cells ͉ small noncoding RNA ͉ transforming growth factor ␤1 D iabetic nephropathy (DN) is the most common cause of kidney failure in patients with diabetes mellitus. The major characteristics of DN include glomerular basement-membrane thickening, mesangial expansion and hypertrophy, and an accumulation of extracellular matrix (ECM) proteins (1). Evidence shows that transforming growth factor ␤1 (TGF-␤) levels are increased under diabetic conditions in renal cells, including mesangial cells (MC), can up-regulate ECM proteins such as collagens (2, 3), and also can promote MC survival and oxidant stress (4).To date, Smad transcription factors have been shown to be the major effectors of TGF-␤ signaling (5, 6). Collagen 1-␣ 1 and -2 (Col1a1 and -2) and other ECM genes are regulated in MC by TGF-␤ via Smads (7,8). The regulation of collagen by TGF-␤ in MC also is mediated by mitogen-activated protein kinases (MAPKs) such as p38 and ERKs (9-11). However, the molecular mechanisms by which TGF-␤ regulates ECM genes still are not understood fully. The collagen gene has E-box elements in the far upstream enhancer region (12, 13). An E-box repressor, ␦EF1, is a key inhibitor of E-cadherin (14) and E2-box transcription factors such as Nkx2.5 (12). Moreover, it is a known repressor of collagen type 1 and type 2 genes in other cells (12, 13), but its role in MC is ...

Akt kinase is activated by transforming growth factor-beta1 (TGF-β) in diabetic kidneys and plays important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells (MC)1–11. However, the mechanisms of Akt activation by TGF-β are not fully understood. Here we show that TGF-β activates Akt in MC by inducing microRNA-216a (miR-216a) and miR-217, both of which target phosphatase and tensin homologue (PTEN). Both these miRs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-β and also by miR-192 via E-box-regulated mechanisms as shown previously3. Akt activation by these miRs also led to MC survival and hypertrophy similar to TGF-β. These studies reveal a mechanism of Akt activation via PTEN downregulation by two miRs regulated by upstream miR-192 and TGF-β. Due to the diversity of PTEN function12, 13, this miR amplifying circuit may play key roles not only in kidney disorders, but also other diseases.

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-27632, conditionally induces an indefinite proliferative state in primary mammalian epithelial cells. These conditionally reprogrammed cells (CRCs) are karyotype-stable and nontumorigenic. Because self-renewal is a recognized property of stem cells, we investigated whether Y-27632 and feeder cells induced a stem-like phenotype. We found that CRCs share characteristics of adult stem cells and exhibit up-regulated expression of α6 and β1 integrins, ΔNp63α, CD44, and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear β-catenin. The induction of CRCs is rapid (occurs within 2 d) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not overexpress the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g., Sox2, Oct4, Nanog, or Klf4). The induction of CRCs is also reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally. Thus, when CRCs from ectocervical epithelium or tracheal epithelium are placed in an air-liquid interface culture system, the cervical cells form a well differentiated stratified squamous epithelium, whereas the tracheal cells form a ciliated airway epithelium. We discuss the diagnostic and therapeutic opportunities afforded by a method that can generate adult stem-like cells in vitro without genetic manipulation.

Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.

The papillomavirus E6 protein binds and directs the ubiquitindependent degradation of the p53 tumor suppressor protein. Independent of this p53-degradative function, however, E6 induces cellular telomerase activity. This increase in enzyme activity reflects E6-enhanced transcription of the human telomerase reverse transcriptase (hTERT) catalytic subunit, but the molecular basis for this transactivation is unknown. In the present study, we demonstrate that E6͞Myc interactions regulate hTERT gene expression. Mad protein, a specific antagonist of Myc, repressed E6-mediated transactivation of the hTERT promoter and this repression was relieved by Myc overexpression. The proximal Myc͞ Max-binding element (E-box) in the hTERT promoter was the major determinant of both E6 and Myc responsiveness in keratinocytes. E6 did not alter Myc protein expression or Myc͞Max association, and the induction of hTERT by Myc͞E6 was independent of Myc phosphorylation at Thr-58͞Ser-62 within the transactivation domain. However, immunoprecipitation studies demonstrated that endogenous Myc protein coprecipitated with E6 protein and chromatin immunoprecipitation analyses demonstrated that both E6 and Myc proteins bound to a minimal 295-bp hTERT promoter. Only the ''high-risk'' E6 proteins bound to the hTERT promoter, consistent with their preferential ability to induce telomerase. The observation that E6 associates with Myc complexes and activates a Myc-responsive gene identifies a mechanism by which this oncogene can modulate cell proliferation and differentiation. T elomerase is a specialized reverse transcriptase that synthesizes repeat DNA sequences at the ends of chromosomes termed telomeres (1). The absence of telomerase activity in most normal human cells results in the progressive shortening of telomeres with each cell division (2-4), eventuating in growth arrest or replicative senescence (3,5). In contrast to most human somatic cells, immortalized and cancer cells contain detectable telomerase activity and consequently maintain their telomere length and proliferative potential (6-9).The telomerase enzyme is a ribonucleoprotein complex comprised of two core subunits, a template RNA subunit [human telomerase RNA (hTR)] (10) and a catalytic protein subunit [human telomerase reverse transcriptase (hTERT)] (11, 12), and subunits important for telomere maintenance and stability (1, 13). Although the hTR template subunit is ubiquitously and equivalently expressed in both normal and tumor tissues (14), the hTERT subunit is selectively expressed in a small subset of normal cells (stem cells), tumor tissues, and tumor-derived cell lines (11,12,15,16), indicating that hTERT is the rate-limiting component of telomerase activity. Indeed, ectopic expression of hTERT alone in telomerase-negative cells is sufficient to restore telomerase activity and induce the immortalization of several primary human cell types (17)(18)(19).The E6 oncoprotein of malignancy-associated human papillomavirus type 16 (HPV-16) has recently been shown to activate telome...

TGF-␤1-induced expression of extracellular matrix (ECM) genes plays a major role in the development of chronic renal diseases such as diabetic nephropathy. Although many key transcription factors are known, mechanisms involving the nuclear chromatin that modulate ECM gene expression remain unclear. Here, we examined the role of epigenetic chromatin marks such as histone H3 lysine methylation (H3Kme) in TGF-␤1-induced gene expression in rat mesangial cells under normal and high-glucose (HG) conditions. TGF-␤1 increased the expression of the ECM-associated genes connective tissue growth factor, collagen-␣1 [⌱], and plasminogen activator inhibitor-1. Increased levels of chromatin marks associated with active genes (H3K4me1, H3K4me2, and H3K4me3), and decreased levels of repressive marks (H3K9me2 and H3K9me3) at these gene promoters accompanied these changes in expression. TGF-␤1 also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to these promoters. SET7/9 gene silencing with siRNAs significantly attenuated TGF-␤1-induced ECM gene expression. Furthermore, a TGF-␤1 antibody not only blocked HG-induced ECM gene expression but also reversed HG-induced changes in promoter H3Kme levels and SET7/9 occupancy. Taken together, these results show the functional role of epigenetic chromatin histone H3Kme in TGF-␤1-mediated ECM gene expression in mesangial cells under normal and HG conditions. Pharmacologic and other therapies that reverse these modifications could have potential renoprotective effects for diabetic nephropathy.

Diabetic nephropathy (DN) is characterized by mesangial cell (MC) expansion and accumulation of extracellular matrixproteins. TGF-␤ is increased in MC under diabetic conditions and in DN and activates key signaling pathways, including the phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway. FoxO transcription factors play roles in cell survival and oxidative stress and are negatively regulated by Akt-mediated phosphorylation. We tested whether phosphorylation-mediated inactivation of FoxO3a by TGF-␤ can mediate MC survival and oxidative stress. TGF-␤ treatment significantly increased levels of p-Akt (activation) and p-FoxO3a (inactivation) in cultured MC. This FoxO3a inactivation was accompanied by significant decreases in the expression of two key FoxO3a target genes, the proapoptotic Bim and antioxidant manganese superoxide dismutase in MC. TGF-␤ treatment triggered the nuclear exclusion of FoxO3a, significantly inhibited FoxO3a transcriptional activity, and markedly protected MC from apoptosis. A PI3K inhibitor blocked these TGF-␤ effects. It is interesting that p-Akt and p-FoxO3A levels also were increased in renal cortical tissues from rats and mice at 2 wk after the induction of diabetes by streptozotocin, thus demonstrating in vivo significance. In summary, TGF-␤ and diabetes can increase FoxO3a phosphorylation and transcriptional inactivation via PI3K/Akt. These new results suggest that Akt/FoxO pathway regulation may be a novel mechanism by which TGF-␤ can induce unopposed MC survival and oxidant stress in early DN, thereby accelerating renal disease.