Lawrence W. Jones

Selective growth of normal adult human urothelial cells in serum-free medium

SummaryA serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human ureter and bladder. Medium HMRI-2 consists of Ham’s MCDB 152 with double the amounts of the essential amino acids in Stock 1, low Ca2+ (0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 μg/ml; insulin, 5 μg/ml; ethanolamine and phosphoethanolamine, 0.1 mM each; hydrocortisone, 2.8×10−6M; and bovine pituitary extract, 126 μg protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype. Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a population doubling time of 40.5±4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca2+ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important in future studies of carcinogenesis.

Unmethylated E‐Cadherin gene expression is significantly associated with metastatic human prostate cancer cells in bone

The concurrent determination of methylation status of E‐cadherin gene and E‐cadherin protein expression remains scant in metastatic prostate cancer cells in bone, the most prevalent site for metastatic growth. Therefore, the study was undertaken to ascertain the methylation status of E‐cadherin gene, a most frequent and known epigenetic mechanism of its regulation, and the protein expression in prostate tissue biopsy specimen.

Differentiation between renal cortex and medulla in the response to hypotension using localized 31p magnetic resonance spectroscopy

Experiments were designed to test the hypothesis that renal medulla is more sensitive to hypoxia than is the cortex. Using the one-dimensional phase encoding technique to perform 31phosphorus magnetic resonance spectroscopy in a perfused porcine kidney preparation, cortex and medulla were differentiated on the basis of the unique resonance at 3 ppm found only in medulla. Hypotension-induced hypoxia reduced total renal oxygen consumption by 60%, and urine flow by 44%. Nonlocalized 31P MRS spectra showed that [ATP]/[Pi] ratio fell by 40%, and intrarenal pH by 0.1 unit. Virtually all of these changes could be accounted for by changes in the renal cortex, where initial [ATP]/[Pi] was higher than in medulla (1.16 vs. 0.68). In medulla [ATP]/[Pi] fell only 29% (n.s. versus control) and pH remained unchanged during hypotension. Thus the cortex appears to be more sensitive to hypoxia in this preparation, and observations fail to support the proposed hypothesis. They are consistent, however, with the greater capacity of medulla for anaerobic glycolysis. Localized 31P MRS provides improved noninvasive metabolic assessment of cold-preserved kidneys.

SRC family kinase FYN promotes the neuroendocrine phenotype and visceral metastasis in advanced prostate cancer

FYN is a SRC family kinase (SFK) that has been shown to be up-regulated in human prostate cancer (PCa) tissues and cell lines. In this study, we observed that FYN is strongly up-regulated in human neuroendocrine PCa (NEPC) tissues and xenografts, as well as cells derived from a NEPC transgenic mouse model. In silico analysis of FYN expression in prostate cancer cell line databases revealed an association with the expression of neuroendocrine (NE) markers such as CHGA, CD44, CD56, and SYP. The loss of FYN abrogated the invasion of PC3 and ARCaPM cells in response to MET receptor ligand HGF. FYN also contributed to the metastatic potential of NEPC cells in two mouse models of visceral metastasis with two different cell lines (PC3 and TRAMPC2-RANKL). The activation of MET appeared to regulate neuroendocrine (NE) features as evidenced by increased expression of NE markers in PC3 cells with HGF. Importantly, the overexpression of FYN protein in DU145 cells was directly correlated with the increase of CHGA. Thus, our data demonstrated that the neuroendocrine differentiation that occurs in PCa cells is, at least in part, regulated by FYN kinase. Understanding the role of FYN in the regulation of NE markers will provide further support for ongoing clinical trials of SFK and MET inhibitors in castration-resistant PCa patients.

Keratin 13 expression reprograms bone and brain metastases of human prostate cancer cells

Lethal progression of prostate cancer metastasis can be improved by developing animal models that recapitulate the clinical conditions. We report here that cytokeratin 13 (KRT13), an intermediate filament protein, plays a directive role in prostate cancer bone, brain, and soft tissue metastases. KRT13 expression was elevated in bone, brain, and soft tissue metastatic prostate cancer cell lines and in primary and metastatic clinical prostate, lung, and breast cancer specimens. When KRT13 expression was determined at a single cell level in primary tumor tissues of 44 prostate cancer cases, KRT13 level predicted bone metastasis and the overall survival of prostate cancer patients. Genetically enforced KRT13 expression in human prostate cancer cell lines drove metastases toward mouse bone, brain and soft tissues through a RANKL-independent mechanism, as KRT13 altered the expression of genes associated with EMT, stemness, neuroendocrine/neuromimicry, osteomimicry, development, and extracellular matrices, but not receptor activator NF-κB ligand (RANKL) signaling networks in prostate cancer cells. Our results suggest new inhibitors targeting RANKL-independent pathways should be developed for the treatment of prostate cancer bone and soft tissue metastases.

Metastasis initiating cells in primary prostate cancer tissues from transurethral resection of the prostate (TURP) predicts castration‐resistant progression and survival of prostate cancer patients

We previously reported that the activation of RANK and c‐Met signaling components in both experimental mouse models and human prostate cancer (PC) specimens predicts bone metastatic potential and PC patient survival. This study addresses whether a population of metastasis‐initiating cells (MICs) known to express a stronger RANKL, phosphorylated c‐Met (p‐c‐Met), and neuropilin‐1 (NRP1) signaling network than bystander or dormant cells (BDCs) can be detected in PC tissues from patients subjected to transurethral resection of the prostate (TURP) for urinary obstruction prior to the diagnosis of PC with or without prior hormonal manipulation, and whether the relative abundance of MICs over BDCs could predict castration‐resistant progression and PC patient survival.

Metabolic Differences in Glutamine Utilization Lead to Metabolic Vulnerabilities in Prostate Cancer

The new oncologic paradigm of precision medicine is focused on identifying metabolic, proteomic, transcriptomic and genomic variabilities in tumors that can be exploited to tailor treatments and improve patient outcomes. Metabolic changes are a hallmark of cancer, and inhibition of metabolic pathways is now a major strategy in medicinal chemistry for targeting cancers. However, non-invasive biomarkers to categorize metabolic subtypes are in short supply. The purpose of this study was to characterize the intracellular and extracellular metabolic profiles of four prostate cancer cell lines with varying degrees of aggressiveness. We observed metabolic differences between the aggressive prostate cancer cell line PC3 and the even more aggressive, metastatic subline PC3M assessed by hyperpolarized in vivo pyruvate studies, nuclear magnetic resonance spectroscopy, and carbon-13 feeding studies. On further examination of the differences between these two cell lines, we found increased glutamine utilization in the metastatic PC3M subline that led directly to sensitivity to glutaminase inhibitor CB-839. Our study supports the theory that metastatic progression increases glutamine utilization and the inhibition of glutaminolysis could have clinical implications.

Fibroblast inhibition of tumor cells may be mediated by TGF-β1

Dear Editor: Several reports show that normal human fibroblasts express an anti-tumor activity in vitro. Fibroblasts, derived from embryonic or neonatal tissue, secrete a soluble anti-tumor substance that is active against a wide range of cell lines established from human and mouse solid tumors (Kirk et al., 1983; Tanaka et al., 1985; Wu et al., 1985; Shirasuna et al., 1988). We monitored the effects of a fibroblast monolayer on the clonal growth of cancer ceils seeded in an overlying agarose gel. Using neonatal human lung fibroblasts, we observe a dose-dependent inhibition of colony forming efficiency with increasing number of fibroblasts seeded (Kirk, 1983). Comparable inhibitions are observed for several different human tumor lines derived from prostate (PC-3, DU-45), bladder (J82), endometrium (HEC-1A) and brain (Hs683t). Further studies using PC-3 cells have shown the inhibitory effect to be a transforming growth factor 81 like activity (TGF-/31). Neutralizing antibody to TGF-/31 caused about a 60% reversal of inhibition in serum-containing co-cultures. A 100% reversal of the inhibitory activity was observed in serum-free conditioned medium concentrated by uhrafiltration (5 kD cut-off). Acid treatment of the serum-free conditioned medium increased its inhibitory activity approximately 20-fold. This enhanced activity was also reversed com-