Measurement and initial characterization of leukocyte telomere length in 474,074 participants in UK Biobank.

Leukocyte telomere length (LTL) is a proposed marker of biological age. Here we report the measurement and initial characterization of LTL in 474,074 participants in UK Biobank. We confirm that older age and male sex associate with shorter LTL, with women on average ~7 years younger in 'biological age' than men. Compared to white Europeans, LTL is markedly longer in African and Chinese ancestries. Older paternal age at birth is associated with longer individual LTL. Higher white cell count is associated with shorter LTL, but proportions of white cell subtypes show weaker associations. Age, ethnicity, sex and white cell count explain ~5.5% of LTL variance. Using paired samples from 1,351 participants taken ~5 years apart, we estimate the within-individual variability in LTL and provide a correction factor for this. This resource provides opportunities to investigate determinants and biomedical consequences of variation in LTL.

Neutral endopeptidase 24.11 loss in metastatic human prostate cancer contributes to androgen-independent progression.

Neutral endopeptidase 24.11 (NEP) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). We report that NEP expression and catalytic activity are lost in vitro in androgen-independent but not androgen-dependent PC cell lines. In vivo, NEP protein expression is commonly decreased in cancer cells of metastatic PC specimens from patients with androgen-independent but not androgen-dependent PC. Overexpression of NEP in androgen-independent PC cells or incubation with recombinant NEP inhibits PC cell growth. Furthermore, in androgen-dependent PC cells, expression of NEP is transcriptionally regulated by androgen and decreases with androgen withdrawal. These data suggest that decreased NEP expression, common in androgen-independent PCs, is facilitated by the elimination of androgens, and that NEP loss plays an important role in the development of androgen-independent PC by allowing PC cells to use mitogenic neuropeptides as an alternate source to androgen in order to stimulate cell proliferation.

Transformation of human kidney proximal tubule cells by ras-containing retroviruses. Implications for tumor progression.

Normal human kidney proximal tubule cells into which a ras oncogene was inserted undergo a series of transformation-related alterations that are characteristic of renal carcinomas. These include changes in morphology, growth potential, anchorage dependence, antigen expression, growth factor production, and chromosomal stability. Further, there are spontaneous progressive alterations in vitro in the karyotype and antigenic profile of the transformed cells. Cytogenetic analyses suggest that alterations of chromosome 21 may play an early and pivotal role in the development of transformed proximal tubule cells.

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase.

The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.

Activation of p300 histone acetyltransferase activity and acetylation of the androgen receptor by bombesin in prostate cancer cells.

Androgen receptor signaling in prostate cancer cells is augmented by the androgen receptor (AR) coactivator p300, which transactivates and acetylates the AR in the presence of dihydrotestosterone (DHT). As prostate cancer (PC) cells progress to androgen independence, AR signaling remains intact, indicating that other factors stimulate AR activities in the absence of androgen. We previously reported that neuropeptide growth factors could transactivate the AR in the presence of very low concentrations of DHT. Here, we examine the involvement of p300 in neuropeptide activation of AR signaling. Transfection of increasing concentrations of p300 in the presence of bombesin into PC-3 cells resulted in a linear increase in AR transactivation, suggesting that p300 acts as a coactivator in neuropeptide-mediated AR transactivation. P300 is endowed with histone acetyltransferase (HAT) activity. Therefore, we examine the effect of bombesin on p300 HAT activity. At 4 h after the addition of bombesin, p300 HAT activity increased 2.0-fold (P<0.01). Incubation with neutral endopeptidase, which degrades bombesin, or bombesin receptor antagonists blocked bombesin-induced p300 HAT activity. To explore the potential signaling pathways involved in bombesin-induced p300 HAT activity, we examined Src and PKCdelta pathways that mediate bombesin signaling. Inhibitors of Src kinase activity or Src kinase siRNA blocked bombesin-induced p300 HAT activity, whereas PKCdelta inhibitors or PKCdelta siRNA significantly increased bombesin-induced p300 HAT activity suggesting that Src kinase and PKCdelta kinase are involved in the regulation of p300 HAT activity. As AR is acetylated in the presence of 100 nM DHT, we next examined whether bombesin-induced p300 HAT activity would result in enhanced AR acetylation. Bombesin-induced AR acetylation at the same motif KLKK observed in DHT-induced acetylation. Elimination of p300 using p300 siRNA reduced AR acetylation, demonstrating that AR acetylation was mediated by p300. AR acetylation results in AR transactivation and the expression of the AR-regulated gene prostate-specific antigen (PSA). Therefore, we examined bombesin-induced AR transactivation and PSA expression in the presence and absence of p300 siRNA and found inhibition of p300 expression reduced bombesin-induced AR transactivation and PSA expression. Together these results demonstrate that bombesin, via Src and PKCdelta signaling pathways, activates p300 HAT activity which leads to enhanced acetylation of AR resulting in increased expression of AR-regulated genes.

Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling.

Neutral endopeptidase 24.11 (NEP, CD10) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). NEP substrates such as bombesin and endothelin-1 induce cell migration. We investigated the mechanisms of NEP regulation of cell migration in PC cells, including regulation of phosphorylation on tyrosine of focal adhesion kinase (FAK). Western analyses and cell migration assays revealed an inverse correlation between NEP expression and the levels of FAK phosphorylation and cell migration in PC cell lines. Constitutively expressed NEP, recombinant NEP, and induced NEP expression using a tetracycline-repressive expression system inhibited bombesin- and endothelin-1-stimulated FAK phosphorylation and cell migration. This results from NEP-induced inhibition of neuropeptide-stimulated association of FAK with cSrc protein. Expression of a mutated catalytically inactive NEP protein also resulted in partial inhibition of FAK phosphorylation and cell migration. Coimmunoprecipitation experiments show that NEP associates with tyrosine-phosphorylated Lyn kinase, which then binds the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in an NEP-Lyn-PI3-K protein complex. This complex competitively blocks FAK-PI3-K interaction, suggesting that NEP protein inhibits cell migration via a protein-protein interaction independent of its catalytic function. These experiments demonstrate that NEP can inhibit FAK phosphorylation on tyrosine and PC cell migration through multiple pathways and suggest that cell migration which contributes to invasion and metastases in PC cells can be regulated by NEP.

Lentiviral vector neutral endopeptidase gene transfer suppresses prostate cancer tumor growth.

Neprilysin (neutral endopeptidase, NEP) is a cell surface peptidase whose expression is lost in approximately 50% of prostate cancers (PC). NEP normally functions to inactivate peptides such as bombesin and endothelin-1, and potentiates the effects of the PTEN tumor suppressor via a direct protein-protein interaction. NEP loss contributes to PC progression. We investigated the therapeutic efficacy of using a lentiviral vector system to restore NEP expression in PC cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP) or green fluorescent protein (L-GFP) were introduced into NEP-deficient 22RV1 PC cells. Cells infected with L-NEP or L-GFP at a multiplicity of infection of 10 demonstrated NEP enzyme activity of 1171.2+/-4.9 and 17.2+/-5.3 pmol/microg/min (P<0.0001), respectively. Cell viability, proliferation and invasion were each significantly inhibited in 22RV1 cells expressing NEP compared with control cells infected with L-GFP (P<0.01). Analysis of known downstream effects of NEP showed NEP-expressing cells exhibiting decreased Akt and focal adhesion kinase phosphorylation and increased PTEN protein expression. Finally, injection of L-NEP into established 22RV1 xenograft tumors significantly inhibited tumor growth (P<0.01). These experiments demonstrate that lentiviral NEP gene transfer is a novel targeted strategy for the treatment of NEP-deficient PC.

Induction of growth factor RNA expression in human malignant melanoma: markers of transformation.

Alteration in the expression of growth factors is widely accepted as being one of several critical defects in the generation of the malignant cell. In the present study, 19 human metastatic melanoma cell lines were compared to 14 normal human foreskin melanocyte cell lines for the production of RNA transcripts specific for 11 different growth factors. Using the extremely sensitive technique of polymerase chain reaction to amplify growth factor-specific complementary DNAs, we analyzed the following: transforming growth factor (TGF) types alpha, beta 1, beta 2, and beta 3, acidic (a) fibroblast growth factor (FGF), basic (b) FGF, FGF-5, keratinocyte growth factor (KGF), HST, and platelet-derived growth factor (PDGF) types A and B. There were clear distinctions among the patterns of growth factor RNA expression by normal melanocytes and malignant melanoma cells. The prototypic melanocyte pattern of expression included TGF beta 1, TGF beta 3, and KGF. A subset of melanocyte cell lines also expressed PDGFA transcripts. In contrast, melanoma cells characteristically expressed RNA transcripts of TGF beta 1, TGF beta 2, TGF beta 3, TGF alpha, bFGF, KGF, and PDGFA. Subsets of melanoma cell lines also expressed aFGF, FGF-5, and PDGFB. The results presented indicated that TGF beta 2, TGF alpha, and bFGF may be particularly important in melanomagenesis and that these, as well as FGF-5, aFGF, and PDGFB, can be used as markers of transformation in this tumor type.

alpha-Interferon down-regulates epidermal growth factor receptors on renal carcinoma cells: relation to cellular responsiveness to the antiproliferative action of alpha-interferon.

The CaKi-I line of renal carcinoma (RC) cells is highly sensitive to the antiproliferative effect of human leukocyte interferon (IFN-alpha). These RC cells express high numbers of cell surface receptors for epidermal growth factor (EGF), and EGF stimulates their proliferation. IFN-alpha blocks EGF-stimulated proliferation of these cells and down-regulates EGF receptors (EGFR) by inhibiting EGFR synthesis. Although EGF stimulates the proliferation of RC cells resistant to the antiproliferative action of IFN-alpha, IFN-alpha treatment does not block the EGF-stimulated proliferation of these cells and has no effect on EGFR expression. Thus, the down-regulation of EGFR is specific for RC cells sensitive to IFN-alpha. While IFN-alpha does not affect the level of total cellular message or total polyadenylated message for the EGFR, IFN-alpha treatment decreases the level of cytoplasmic EGFR message. Analysis of polysome distribution of cellular mRNAs indicates that IFN-alpha treatment results in an accumulation of EGFR mRNA in lighter polysome fractions, consistent with a partial block in translational elongation. Thus, IFN-alpha regulates the expression of EGFR and possibly other growth-related proteins by post-transcriptional mechanisms, which may play an important part in the complex inhibitory action of IFN-alpha on RC proliferation.

Antiproliferative and antitumor effects of alpha-interferon in renal cell carcinomas: correlation with the expression of a kidney-associated differentiation glycoprotein.

Human leukocyte alpha-interferon (IFN-alpha) has significant antitumor activity in advanced renal cell carcinoma (RC), with approximately 15% (range, 5 to 29%) of patients subjected to IFN-alpha therapy exhibiting a major objective response. We assayed 16 RC cell lines for intrinsic sensitivity to the growth-inhibitory effects of recombinant IFN-alpha. Similar to results observed in patients, cultured RCs could be divided into those that are inhibited by IFN-alpha and those that are not. In addition, the IFN-alpha-sensitive or -resistant phenotype of cultured RCs was correlated with surface expression of six unrelated kidney-associated differentiation antigens. The expression of one antigen, a Mr 160,000 glycoprotein (gp160), was found to correlate with resistance to IFN-alpha. Proliferation of seven RC cell lines expressing gp160 (gp160+) was not significantly inhibited by IFN-alpha at concentrations as high as 3000 units/ml. In contrast, proliferation of eight of nine RC cell lines lacking expression of gp160 (gp160-) was markedly inhibited by IFN-alpha. The effect of IFN-alpha on gp160+ and gp160- RC xenografts in nu/nu mice was examined. In separate experiments, two gp160+ RC cell lines and five gp160- RC cell lines were injected s.c. into nu/nu mice; one half of the mice were subsequently treated with 10(6) units of IFN-alpha i.p. 3 times a wk, and one half received no IFN-alpha. Tumors appeared at the sites of inoculation in all mice given injections of gp160+ RC cell lines within 10 to 25 days regardless of INF-alpha therapy. Mice given injections of gp160- RC cell lines, but not receiving IFN-alpha, also formed tumors. In contrast, gp160- RC cell lines injected into mice that were treated with IFN-alpha exhibited a marked sensitivity, as demonstrated by either no tumor formation or delayed tumor formation. We conclude that the absence of gp160 expression by RCs may be predictive of sensitivity to the antitumor effects of IFN-alpha and, thus, provide a basis for identifying IFN-responsive patients.

Reduced levels of retinyl esters and vitamin A in human renal cancers.

Clinical and preclinical studies suggest that retinoids can inhibit the growth of a small percentage of human renal cancers (RCs), although the majority of RCs both in vitro and in vivo are retinoid resistant. Our recent studies indicate that the metabolism of retinol to retinyl esters is greatly reduced in human carcinoma cell lines of the oral cavity, skin, and breast as compared with their normal epithelial counterparts, suggesting that human carcinoma cells are retinoid deficient relative to normal epithelial cells. We considered whether retinoid resistance in RCs was related to an abnormality in retinoid metabolism. The metabolism of [3H]retinol and of [3H]retinoic acid (RA) was examined in RC cell lines and normal human kidney (NK) epithelial cells cultured in media, in RA, or in RA plus IFN-alpha. The expression of LRAT (lecithin:retinol acyltransferase) was assessed by Northern and Western analysis. Retinol and retinyl ester levels were determined in tissue samples of normal human kidney and renal cell carcinoma. NK cells esterified all of the 50 nM [3H]retinol in which they were cultured. In contrast, six of the seven RC cell lines metabolized only trace amounts of [3H]retinol to [3H]retinyl esters. Consistent with this relative lack of [3H]retinol esterification by the tumor cells, the tumor cells exhibited LRAT transcripts of aberrantly low sizes relative to those in normal epithelial cells. Moreover, the NK cells expressed abundant levels of LRAT protein by Western analysis, whereas the RC cells did not express LRAT protein. When samples of human kidney tumor tissue were compared with samples of normal kidney tissue from patients who had undergone surgery for primary RC, the normal kidney tissues contained much higher levels of retinol and retinyl esters (approximately 0.5-2 microg/gram wet weight) than the tumor tissues in all seven patients examined. Culture of the RC lines in IFN-alpha plus all-trans-RA, a combination therapy used clinically, resulted in higher intracellular levels of [3H]retinol and [3H]retinyl esters. The metabolism of [3H]RA was also examined in these RC lines versus NK cells. Although the NK epithelial cells metabolized [3H]RA, the majority of the RC lines metabolized [3H]RA at a much slower rate. Most of the RC lines metabolized only 10-30% of the 50 nM [3H]RA over 6 h of culture. These data indicate that RCs both in vitro and in vivo are retinol and retinyl ester deficient relative to the normal human kidney, and they suggest that the aberrant differentiation of the neoplastic renal cells results in part from a defect in retinoid metabolism.

Neutral endopeptidase promotes phorbol ester-induced apoptosis in prostate cancer cells by inhibiting neuropeptide-induced protein kinase C delta degradation.

Phorbol esters induce apoptosis in androgen-sensitive LNCaP cells, which express neutral endopeptidase (NEP), but not in androgen-independent prostate cancer (PC) cells, which lack NEP expression. We investigated the role of NEP in PC cell susceptibility to 12-O-tetradecanoylphorbol-13-acetate (TPA). Western analysis showed that expression of NEP and protein kinase Cdelta (PKCdelta) correlated with PC cell sensitivity to TPA-induced growth arrest and apoptosis in LNCaP cells and in TSU-Prl cells expressing an inducible wild-type NEP protein. Inhibition of NEP enzyme activity using the specific NEP inhibitor CGS24592, or inhibition of PKCdelta using Rottlerin at concentrations that inhibit PKCdelta but not PKCalpha, significantly inhibited TPA-induced growth inhibition and cell death. Furthermore, pulse-chase experiments showed PKCdelta is stabilized in LNCaP cells and in TSU-Pr1 cells overexpressing wild-type NEP compared with PC cells lacking NEP expression. This results from NEP inactivation of its neuropeptide substrates (bombesin and endothelin-1), which in the absence of NEP stimulate cSrc kinase activity and induce rapid degradation of PKCdelta protein. These results indicate that expression of enzymatically active NEP by PC cells is necessary for TPA-induced apoptosis, and that NEP inhibits neuropeptide-induced, cSrc-mediated PKCdelta degradation.

Phase II trial of suramin in patients with advanced renal cell carcinoma: treatment results, pharmacokinetics, and tumor growth factor expression.

Twenty-six patients with advanced renal cell carcinoma were treated with suramin administered by continuous infusion, with dosing determined by a nomogram. One patient achieved a partial response and five patients achieved a minor response or had stable disease for > 3 months. Toxicities included an immune-mediated thrombocytopenia in one patient and Staphylococcus sepsis that was not associated with neutropenia in five patients. Pharmacokinetic parameters were determined by the ADAPT II MAP-Bayesian parameter estimation program. Patient data were fit using a two-compartment open model and first-order rate elimination. This showed a wide interpatient variation in time to target level (median, 13.8 days), volume of distribution (median, 15.2 liters/m2), and t1/2-beta (median, 20.6 days). The patients who achieved a partial response, minor response, or stable disease had a slower elimination rate of suramin, compared to patients with progressive disease. Tumor specimens were obtained prior to therapy and were analyzed for the production of five different growth factor-specific RNA transcripts. These included transforming growth factor alpha, acidic fibroblast growth factor, basic fibroblast growth factor, and platelet-derived growth factor types A and B. No difference in the pattern of growth factor expression was seen in tumors of responding and nonresponding patients. Suramin does not have significant antitumor activity in renal cell carcinoma. The wide variability in pharmacokinetics suggests that individual dosing should be used in future trials of suramin for treatment for other malignancies. Pertinent corollary studies of tumor biology and clinical pharmacology should be included whenever possible in clinical trials in patients with renal cell carcinoma.

Neutral endopeptidase inhibits neuropeptide-mediated transactivation of the insulin-like growth factor receptor-Akt cell survival pathway.

G-protein coupled receptor (GPCR) agonists such as neuropeptides activate the insulin-like growth factor-1 receptor (IGF-IR) or the serine-threonine protein kinase Akt, suggesting that neuropeptides-GPCR signaling can cross-communicate with IGF-IR-Akt signaling pathways. Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase that cleaves and inactivates the neuropeptides endothelin-1 (ET-1) and bombesin, which are implicated in progression to androgen-independent prostate cancer (PC). We investigated the mechanisms of NEP regulation of neuropeptide-mediated cell survival in PC cells, including whether neuropeptide substrates of NEP induce phosphorylations of IGF-IR and Akt in PC cells. Western analyses revealed ET-1 and bombesin treatment induced phosphorylation of IGF-IRbeta and Akt independent of IGF-I in TSU-Pr1, DU145, and PC-3 PC cells, which lack NEP expression, but not in NEP-expressing LNCaP cells. Recombinant NEP and induced NEP expression in TSU-Pr1 cells using a tetracycline-repressive expression system inhibited ET-1-mediated phosphorylation of IGF-IRbeta and Akt, and blocked the protective effects of ET-1 against apoptosis induced by serum starvation. Incubation of TSU-Pr1 cells with specific kinase inhibitors together with ET-1 or bombesin showed that IGF-IR activation is required for neuropeptide-induced Akt phosphorylation, and that neuropeptide-induced Akt activation is predominantly mediated by Src and phosphatidylinositol 3-kinase but not by mitogen-activated protein kinase or protein kinase C. These data show that the neuropeptides ET-1 and bombesin stimulate ligand-independent activation of the IGF-IR, which results in Akt activation, and that this cross-communication between GPCR and IGF-IR signaling is inhibited by NEP.

PO-43 - Differential coagulation factor expression in neuroendocrine prostate cancer (PC), metastatic castrate-resistant PC, and localized prostatic adenocarcinoma.

INTRODUCTION: Neuroendocrine prostate cancer (NEPC) is an aggressive late-stage variant of PC that is often androgen-receptor negative. Most clinicians believe the VTE rate with NEPC is higher than with standard metastatic castration-resistant PC (mCRPC), but NEPC tends to present with bulkier visceral disease and include platinum chemotherapy unlike standard PC. In many solid tumors, a more aggressive phenotype correlates with increased VTE risk and elevated expression of coagulation factors. We previously reported on the differential expression of thrombin and tissue factor (TF) in NEPC versus localized PC and benign prostate tissue with a small NEPC cohort (N=7), which showed overexpression of prothrombin and reduced expression of TF in NEPC. AIM: To compare the expression of coagulation factors of NEPC vs mCRPC (and localized PC control) in an expanded datase. MATERIALS AND METHODS: Fresh frozen tissue biopsies were collected and separated into three cohorts based on pathology: localized PC (N=68), standard mCRPC (N=32), and NEPC (N=21). RNA was isolated and next generation paired-end mRNA sequencing was performed on Illumina Sequencers. F2 Prothrombin (F2), tissue factor (F3), carboxypeptidase (CPB2), fibrinogen (FGG, FGA), PAR-1 (F2R), and PAR-2 (F2RL1) were compared by Wilcoxon tests. RESULTS: Prothrombin had significantly higher expression in NEPC versus standard mCRPC (p <0.001). NEPC trended towards higher expression of CPB2 (p=0.1) and lower expression of F3 (p=0.23) and F2RL1 (p=0.14) compared to mCRPC. Compared to localized PC, both types of advanced disease (NEPC and mCRPC) overexpressed F2, FGA, FGB, and CPB2 (p<0.001) and had decreased expression of F3 and F2RL1 (p <0.001). CONCLUSIONS: Prothrombin is reliably overexpressed in NEPC vs mCRPC and localized PC. Advanced disease (regardless of subtype) is associated with significantly higher expression of prothrombin, fibrinogen, and carboxypeptidase and lower expression of TF and PAR-2. It is possible that there may be PC-specific differences with aggressive disease associated with the thrombin axis vs the more common TF/PAR2 axis commonly seen in other advanced solid tumors. Further research is required to understand these differences in biology and resulting thrombotic and hemostatic outcomes.

Malignant transformation of human melanocytes: induction of a complete melanoma phenotype and genotype.

Human melanoma provides a model to study malignant transformation and tumor progression. Expression of ras oncogenes in cultured normal human diploid melanocytes has induced a subset of phenotypic traits that are characteristic of malignant melanoma cells, including altered morphology, anchorage independence, induction of class II MHC antigens, up-regulation of the ganglioside GD3, and chromosomal abnormalities. However, other characteristics of melanoma, such as loss of expression of adenosine deaminase-binding protein and tumorigenicity, were not observed. We report here that melanocytes infected with a retrovirus containing the viral Ha-ras oncogene underwent complete transformation, acquiring all phenotypic characteristics of malignant melanomas observed in vivo. Transformation occurred in a sequential manner and was associated with spontaneous chromosomal instability. Cytogenetic analysis of transformed melanocytes indicated that the earliest structural chromosomal abnormalities were isochromosomes 6p and 9q followed by complete loss of chromosome 1p, all common karyotypic abnormalities described in human melanomas. The findings suggest that these chromosome regions which are deleted or relatively deficient may contain genes that are critical for the initiation and progression of the melanoma phenotype.

Isochromosome 12p in non-seminoma cell lines: karyologic amplification of c-ki-ras2 without point-mutational activation.

We examined eight human germ cell cancer lines (GCCLs) for cytogenetic abnormalities and found an isochromosome 12p, i(12p), marker in all seven male nonseminoma GCCLs, but not in the single female teratocarcinoma cell line. Southern blot analysis of these cell lines showed increased copy number for c-ki-ras2, a gene located on 12p, in all the male GCCLs. The comparison of Southern blot analysis for a restriction fragment length polymorphism (RFLP) probe localized to 12p to a probe for int-1, which maps to 12q, indicates that the increased copy number for c-ki-ras2 is primarily from the greater numbers of 12p relative to 12q. Although Northern analysis revealed enhanced mRNA expression for c-ki-ras2 in the GCCLs with an i(12p), hybridization of specific end-labelled oligonucleotides to the polymerase chain reaction products of c-ki-ras2 codons 12, 13, or 61 did not identify c-ki-ras2 mutations of these codons in these cells. Thus, c-ki-ras2 activation through point mutation is an infrequent event in GCCLs. These data further suggest that increased 12p copy number is a common event in the transformation process leading to male germ cell cancer. We conclude that determination of 12p copy number by cytogenetic analysis or Southern blotting is useful in the diagnostic evaluation of human germ cell cancer.

Elevated expression of protein tyrosine kinase c-Yes, but not c-Src, in human malignant melanoma.

The c-yes proto-oncogene encodes a protein tyrosine kinase, p62c-yes (c-Yes) that belongs to the Src family of non-receptor type protein tyrosine kinases. We compared the levels of c-Yes kinase activity and protein by immune complex kinase assays and immune blot analysis in 20 human melanoma and 10 human melanocyte cell lines. Results show that the average kinase activity of c-Yes in most melanoma cell lines is 5-10-fold higher than that in melanocyte cell lines. The protein level of c-Yes in these melanoma cell lines is correspondingly higher than that in melanocytes. The increase in c-Yes kinase activity is most likely attributable to the elevated protein level because single-strand conformational polymorphism of all structural and functional domains detected no mutations in any of the c-yes coding regions. Subcellular fractionation analysis indicated that c-Yes localizes to the plasma membrane, perinuclear and cytosolic compartments while c-Src predominantly associates with plasma membranes. In melanoma cells in which an elevated level of c-Yes is observed, a protein of 39 kD is heavily phosphorylated on tyrosine. This protein is only observed in melanoma cells and not in melanocytes, suggesting a perturbed signaling pathway in melanoma cells that results in abnormal tyrosine phosphorylation of cellular proteins. These data suggest that derangement of expression of the c-Yes tyrosine kinase may have a role in the malignant progression of the human melanocyte.

Transformation of human kidney proximal tubule cells by a src-containing retrovirus.

Analysis of the cell-surface antigenic phenotypes of normal and malignant renal cells demonstrates that approximately 90% of cultured normal human kidney cells and virtually all renal cell carcinomas (RCC) derive from the proximal tubule (PT) cell of the nephron. In the present study, the v-src oncogene was introduced into cultured normal human PT cells, and the effects of the v-src-encoded protein (pp60v-src) on the biologic and genetic phenotype of these cells were determined. V-src-containing PT cells underwent a series of phenotypic changes characteristic of renal cells in vivo. These included (i) alterations in morphology, (ii) an increase in proliferative capacity, (iii) loss of contact inhibition, (iv) immortalization and (v) tumorigenicity. Moreover, v-src-infected PT cells developed non-random clonal karyo-typic abnormalities which are commonly observed in RCCs, including a deletion of chromosomal region 3p14-21, one of the most frequent deletions observed in human renal tumors. These results indicate that pp60v-src can initiate a complex process leading to the transformation of PT cells. This process includes the induction of genetic instability. Finally, these data provide experimental evidence corroborating cytogenetic and molecular data that a deletion of genes on chromosome 3p is a critical event in the transformation of the human renal cell.