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D F Jarrard

Publications and source records attributed to D F Jarrard.

At least 19 recordsLinked to original sources
PubMed2001

Role of cyclin-dependent kinase inhibitors in the growth arrest at senescence in human prostate epithelial and uroepithelial cells.

Cellular senescence has been proposed to be an in vitro and in vivo block that cells must overcome in order to immortalize and become tumorigenic. To characterize these pathways, we focused on changes in the cyclin-dependent kinase inhibitors and their binding partners that underlie the cell cycle arrest at senescence. As a model, we utilized normal human prostate epithelial cell (HPEC) and human uroepithelial cell (HUC) cultures. After 30-40 population doublings cells became growth-arrested in G0/1 with a threefold decrease in Cdk2-associated activity, a point defined as pre-senescence. Temporally following this growth arrest, the cells develop a senescence morphology and express senescence-associated beta-galactosidase (SA-beta-gal). Levels of p16(INK4a) and p57(KIP2) rise in HUCs during progressive passages, whereas only p16 increases in HPEC cultures. The induced expression of p57, similar to p16, produces a senescent-like phenotype. pRB, cyclin D, p19(INK4d) and p27(KIP1) decrease in both cell types. We find that p53, p21(CIP1) and p15(INK4b) are transiently elevated in HPECs and HUCs at the pre-senescent growth arrest, then return to low proliferating levels at terminal senescence. Analysis of p53, p21(CIP1), p15(INK4b), p16(INK4a), and p57(KIP2) reveals altered expression in immortalized, non-tumorigenic HPV16 E6 and E7 prostate lines and in tumorigenic prostate cancer cells. These results indicate: (i) the existence of a subset of growth inhibiting genes elevated at the onset of the senescence, (ii) a distinct class of genes involved in the maintenance of senescence, and (iii) the frequent inactivation of these pathways during immortalization.

Adult↗
PubMed2001

Hand-assisted laparoscopic radical nephrectomy: comparison to open radical nephrectomy.

OBJECTIVES: Hand-assisted laparoscopic surgery is easier to learn than standard laparoscopy and simplifies intact specimen removal. We present our experience performing hand-assisted laparoscopic radical nephrectomy (HALRN) and compare it with contemporary open radical nephrectomy performed at our institution. METHODS: We performed 18 HALRNs for renal tumors ranging in size from 2 to 11 cm (average 4.5). Patients ranged in age from 40 to 83 years (average 62.9). All patients underwent HALRN with intact removal through a 7 to 8-cm vertical midline incision through an impermeable wound protector. Two or three working ports were used. We retrospectively compared our results with the results of 18 open radical nephrectomies performed during the same period, with the patients matched for age, body mass index, and American Society of Anesthesiologists' score. RESULTS: In the HALRN group, the average operating room time was 220.5 minutes, average length of stay 3.9 days, average time to return to normal activity 15.8 days, and average time to return to work 26.8 days. The median time to return to 100% normal was 28.0 days. No conversions or re-explorations were necessary in the HALRN series. The final pathologic examination revealed renal cell carcinoma in 15, oncocytoma in 1, angiomyolipoma in 1, and a complex cyst in 1. At a maximum of 48 months of follow-up (average 12.2), no recurrences were identified. Three deaths occurred in the series; 2 patients died with no evidence of disease and 1 patient died of metastatic disease (the nephrectomy was palliative). In the open group, the average operating room time was 117.8 minutes, average length of stay 5.1 days, average time to return to normal activity 23.5 days, and average time to return to work 52.2 days. The median time to return to 100% normal was 150 days, with 3 patients never returning to 100% normal. CONCLUSIONS: Our series demonstrated that HALRN is a safe, effective, minimally invasive option for treating renal cell carcinoma and provides a shorter hospital stay (P = 0.02), earlier return to work (P = 0.04), and earlier return to 100% normal (P = 0.0002) than open radical nephrectomy.

Adenoma, Oxyphilic↗
PubMed2000

Different combinations of genetic/epigenetic alterations inactivate the p53 and pRb pathways in invasive human bladder cancers.

Inactivation of both the pRb (pRb-cyclin D1/cyclin-dependent kinase 4/6-p16) and p53 (p53-p21(WAF1)-p14(ARF)) pathways is thought to be essential for immortalization in vitro and malignant transformation in vivo. We identified different combinations of pRb and p53 pathway alterations in 12 invasive transitional cell carcinomas (TCCs) and addressed the functional significance of the different combinations observed. Results showed four combinations of alterations including -pRb/-p53 (ie., pRb inactivated in the pRb pathway and p53 inactivated in the p53 pathway; four TCCs), -p16/-p53 (four TCCs), -p16/-p21(WAF1) (one TCC), and -p16/ -p14(ARF) (two TCCs). These groups include two new combinations (ie., -p16/-p53 and -p16/-p21(WAF1)) not reported previously for TCCs. An alteration in the key components of the p53 pathway was not detected in one invasive TCC that had inactivated p16. Note that all four TCCs with inactivated pRb had mutant p53; thus, the combinations of -pRb/ -p21(WAF1) and -pRb/-p14(ARF) were not observed. Only two of eight TCCs with altered p16 had concomitant p14(ARF) loss, demonstrating that simultaneous inactivation of these two 9p21INK4a tumor suppressor genes is not obligatory. To determine the biological phenotypes of TCCs with different combinations of pRb and p53 pathway alterations, their downstream responses to gamma radiation were studied in vitro. As expected, none of eight TCCs with mutant p53 responded to gamma radiation by elevation of p53, p21(WAF1), or mdm2 or by cell cycle arrest. Only two of four TCCs with wild-type p53 and wild-type pRb (the combination of -p16/-p14(ARF)) showed normal downstream responses to gamma radiation and underwent cell cycle arrest. Two TCCs with wild-type pRb and wild-type p53 (the combination of -pl6/-p21(WAF1) and one TCC with -p16) failed to show cell cycle arrest in response to radiation. This was attributed to the absence of p21(WAF1) in one TCC. In summary, these data support a model of invasive bladder cancer pathogenesis in which both the pRb and p53 pathways are usually inactivated and the biology of the tumor is impacted by the mechanism of their inactivations.

Blotting, Northern↗
PubMed2000

Methylation of the androgen receptor minimal promoter silences transcription in human prostate cancer.

Advanced hormone-independent prostate cancer is characterized by a significant loss of androgen receptor (AR) expression in 20-30% of the tumors. The transcriptional block underlying this phenomenon is not known, but we have proposed that methylation of CpG sites in the AR promoter may reversibly inactivate transcription of the AR (D. F. Jarrard et al, Cancer Res., 58: 5310-5314, 1998). In this study, detailed methylation analysis using bisulfite sequencing was performed on a series of AR expression-positive and -negative prostate cancer cells. We found that methylation of several consensus sequences in the AR promoter (from -131 to -121 and +44 to +54) are tightly linked to the loss of AR expression in metastatic hormone-independent prostate cancer cell lines. These consensus sites of methylation correlate with the minimal promoter region critical for AR transcription. In human tissues, no methylation was demonstrated in normal or primary prostate cancers that express the AR. Four of 15 tumors obtained from men who had died from hormone-independent prostate cancer demonstrated a significant loss of AR expression immunohistochemically and two (50%) of these AR-negative tumors contained AR methylation. We conclude that the AR promoter contains specific CpG methylation hot spots that are markers for gene silencing. Furthermore, AR methylation may represent a phenotype important in the development of hormone independence in a subset of advanced prostate cancer in which AR expression is lost. The finding of AR methylation also represents the first report of aberrant methylation on an X-linked gene associated with a somatic male cancer.

3' Untranslated Regions↗
PubMed2000

Screening hypermethylated regions by methylation-sensitive single-strand conformational polymorphism.

DNA methylation is an important epigenetic modification that alters transcription in those genes containing CpG islands. In this report we demonstrate that the familiar technique single-strand conformation polymorphism (SSCP) can be easily applied to bisulfite-treated DNA to detect methylation differences over large ( approximately 250 bp) CG-enriched regions. In the methylation-sensitive SSCP (Ms-SSCP) technique, sodium bisulfite modification of DNA converts unmethylated cytosine to uracil under conditions in which 5-methylcytosine remains unaltered. Amplification of these regions to be tested is then performed using primers specific for bisulfite-treated DNA. The resulting products are then subjected to nondenaturing gel analysis. Based on differential band mobility, Ms-SSCP is able to sensitively detect alterations in methylation density and determine if unmethylated alleles are present in a sample. Ms-SSCP is a rapid and simple technique for screening multiple samples for methylation changes. It has several advantages over existing techniques, including the utilization of nanogram amounts of DNA, the avoidance of difficulties in sequencing CG-enriched regions, and the screening of multiple sites for methylation across large regions of DNA.

DNA↗
PubMed1999

p16/pRb pathway alterations are required for bypassing senescence in human prostate epithelial cells.

The cell cycle regulatory genes p16/CDKN2 and RB are frequently deleted in prostate cancers. In this study, we examined the role of alterations in p16 and pRb during growth, senescence, and immortalization in vitro of human prostate epithelial cells (HPECs). HPECs are established from normal prostate tissues and cultured on collagen-coated dishes. Our results show that p16 is reproducibly elevated at senescence in HPECs. HPECs are immortalized using human papilloma virus 16 E6 and/or E7 as molecular tools to inactivate p53 and/or pRb, respectively. Immortalization occurs infrequently in this system and only after a latent period during which additional genetic/epigenetic changes are thought to occur. Notably, all of the E6-immortalized HPEC lines but none of the E7 lines show inactivation of p16/CDKN2 (by deletion, methylation, or mutation) in association with immortalization. In contrast, E7 lines, in which pRb function is abrogated by E7 binding, retain the high levels of p16 observed at senescence. Thus, all lines show either a p16 or pRb inactivation. Analysis of six independent lines from metastatic prostate cancers reveals a similar loss of either p16 or pRb. Comparative genomic hybridization of HPECs shows that gains of chromosomes 5q, 8q, and 20 are nonrandomly associated with bypassing senescence (probability = 0.95). These results suggest that high levels of the cyclin-dependent kinase inhibitor p16 mediate senescence G1 arrest in HPECs and that bypassing this block by a p16/pRb pathway alteration is required for immortalization in vitro and possibly tumorigenesis in vivo. Our results further indicate that inactivation of the p16/pRb pathway alone is not sufficient to immortalize HPECs and that additional genetic alterations are required for this process.

Aged↗
PubMed1998

Methylation of the androgen receptor promoter CpG island is associated with loss of androgen receptor expression in prostate cancer cells.

Androgen-independent metastatic prostate cancer is characterized by a heterogeneous loss of androgen receptor (AR) expression among tumor cells. In this study, we evaluate DNA hypermethylation as a potential transcriptional regulatory mechanism in AR-negative prostate cancer cell lines. Nucleotide sequence analysis demonstrates an approximately 15-kb CpG island in the AR gene that encompasses the transcription start site and exon 1. Using Southern blotting with methylation-sensitive restriction enzymes and methylation-specific PCR, we find aberrant methylation in the AR expression-negative cell lines Du145, DuPro, TSU-PR1, and PPC1. Incomplete methylation in the AR CpG island is also seen in normal female breast and ovarian tissues consistent with the inactivation of one X chromosome by hypermethylation. In contrast, prostate cancer cell lines LNCaP and PC3 express AR and are unmethylated. Normal prostate epithelial cell strains demonstrate no methylation. Exposure of AR-negative prostate cancer cell lines to 5-aza-2' deoxycytidine, a demethylating agent, induces the reexpression of AR RNA in DuPro and TSU-PR1. This reexpression is associated with a demethylation of this region. Prostate-specific antigen, an androgen-responsive gene, is also specifically induced in these lines after AR reexpression. Therefore, in vitro DNA methylation of the 5' CpG AR island may be associated with the loss of AR expression. Furthermore, our results demonstrate that treatment with demethylating agents may engender the reexpression and function of the androgen receptor in AR-negative cell lines.

CpG Islands↗
PubMed1998

Overcoming cellular senescence in human cancer pathogenesis.

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and -3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P = 0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11-q12 (0.99) and +8p22-pter (0.94) in the immortal muscle invasive TCCs, and of -9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13-p14. In this study, four of six myoinvasive TCCs also showed TP53 mutation that associated well with genome instability (P = 0.001), as previously hypothesized. Notably, TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P = 0.04) than was p16 or pRb alteration (P = 0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11-q12, -3p13-p14, or -8p21-pter.

Adult↗
PubMed1997

Methylation of the 5' CpG island of the endothelin B receptor gene is common in human prostate cancer.

Production of the potent vasoconstrictor endothelin-1 (ET-1) by human prostate cancer cells accompanies prostate cancer progression in vivo. The predominant endothelin receptor expressed by normal prostate epithelium, ETB, is not expressed by any of the established human prostate cancer cell lines, and ETB binding is decreased on prostate cancer tissues. ETB, which may mediate ET-1 clearance and may inhibit ET-1 secretion, is encoded by a gene that contains a 5' CpG island encompassing the transcriptional regulatory region. We examined this regulatory region of the ETB receptor gene (EDNRB) to determine whether hypermethylation of cytidine nucleotides accompanies decreased ETB expression in human prostate cancer. We found somatic methylation of CpG island sequences in EDNRB in 5 of 5 human prostate cancer cell lines, 15 of 21 primary prostate cancer tissues, and 8 of 14 prostate cancer metastases (70% of samples overall). Normal tissues contained only unmethylated EDNRB. Treatment of human prostatic carcinoma cell line cultures with 5-azacytidine induced ETB mRNA expression, suggesting that CpG island methylation changes might accompany the apparent transcriptional silencing of EDNRB in vivo.

Dinucleoside Phosphates↗
PubMed1997

The cadherin cell-cell adhesion pathway in prostate cancer progression.

Cadherins are a family of calcium-dependent cell-cell adhesion molecules involved in cell-cell aggregation and morphoregulatory cell function. Dysfunction of the cadherin pathway is involved in tumour invasiveness and disease progression for a variety of carcinomas. E-cadherin is a prognostic marker in prostatic cancer, based on the correlation of the grade of E-cadherin expression and tumour grade, stage, metastasis and survival, as well as recurrence after radical prostatectomy. P-cadherin was shown to be lost in all prostatic cancers, although this most likely reflects loss of the basal cell population rather than a transcriptional down-regulation, suggesting that loss of P-cadherin expression is an early event in the tumorigenesis of prostatic carcinomas. Catenins, particularly alpha-catenin, also play an important role in the dysfunction of the cell adhesion complex. Mechanisms of inactivation of the cadherin-catenin pathway include LOH, gene deletions and gene promoter hypermethylation. Therapeutic strategies have been investigated in tumour models, i.e. the use of demethylating agents for the hypermethylated promoter region of E-cadherin or gene transfer in PC-3 cells with homozygous deletion of the alpha-catenin gene. The complexity of neoplastic changes cannot be explained by alterations of cell adhesion molecules alone; but as demonstrated, cadherins and catenins play an important role in this process.

Cadherins↗
PubMed1997

Deletional, mutational, and methylation analyses of CDKN2 (p16/MTS1) in primary and metastatic prostate cancer.

The tumor suppressor gene CDKN2 (p16/MTS1) resides on chromosome 9p21 and encodes a 16 kDa inhibitor of the cyclin-dependent kinases. Inactivation of CDKN2 by homozygous deletion, point mutation, and recently described aberrant methylation in the 5' promoter region may increase progression through the cell cycle in tumors. In this study, we examine the CDKN2 gene for the presence of inactivating alterations in human prostate cancer. Sequence analysis of cell lines revealed no mutation in LNCaP, PC3, and TSU-PR1 and a missense mutation, GAC-->TAC (asp to tyr), in exon 2 of the DU145 cell line at codon 76. No mutations were identified in three primary prostate cancers or in seven lymph node metastases. Loss of heterozygosity (LOH) was analyzed by analysis of microsatellite markers in the vicinity of the CDKN2 gene. LOH was detected in 12 (20%) of 60 primary tumors at one or more loci and in 13 (46%) of 28 metastases. Methylation analysis of the CpG-rich promoter region revealed a dense methylation of CDKN2 in cell lines PC3, PPC1, and TSU-PR1, and this was found to correlate with a lack of mRNA expression by reverse transcription-polymerase chain reaction. A demethylating agent, 5-aza-2'-deoxycytidine, induced reexpression when cells were exposed in vitro. DU145 and LNCaP expressed the CDKN2 transcript and were unmethylated in the promoter region. Three of twenty-four (13%) primary prostate cancers and 1 of 12 metastatic tumors demonstrated promoter methylation. No normal prostate tissues were methylated at the CDKN2 gene promoter. One tumor was found to contain concomitant LOH and promoter methylation indicative of biallelic inactivation. A comprehensive analysis of CDKN2 in prostate cancer reveals that point mutations are infrequent, but gene deletion and methylation combine to inactivate CDKN2 in a subset of tumors. Moreover, alterations in this gene may represent a late event in prostate cancer progression.

Aged↗
PubMed1997

P-Cadherin is a basal cell-specific epithelial marker that is not expressed in prostate cancer.

P-Cadherin is a member of the cadherin family of cell surface glycoproteins that mediate Ca2+-dependent cell-cell adhesion and is expressed in a differential fashion in normal epithelial tissues. The expression of P-cadherin in human prostate cancer development has not been investigated previously. By immunohistochemistry, we show that P-cadherin expression is restricted to the cell-cell border of basal epithelial cells in 30 normal prostate samples. This staining is down-regulated in prostatic intraepithelial neoplasia and is absent in all 25 of the well to poorly differentiated prostate cancer specimens analyzed. To examine potential P-cadherin-regulatory elements, we sequenced the 5'-flanking region of this gene. Similar to the mouse gene, the human P-cadherin promoter is TATA-less, contains an Sp-1 binding site and, analogous to the human E-cadherin sequence, demonstrates a GC-rich region characteristic of a CpG island. Cytosine methylation of this region occurs in P-cadherin-negative prostate cancer cell lines but not in cell lines expressing this gene. In vivo, a lack of expression in 12 clinical prostate cancer specimens is not associated with methylation of the P-cadherin promoter. These results demonstrate that the expression of the basal cell marker P-cadherin is lost in prostate cancer development and that in vivo mechanisms other than cytosine methylation regulate this consistent loss of expression.

Animals↗
PubMed1996

DNA methylation, molecular genetic, and linkage studies in prostate cancer.

Molecular biologic studies have now identified a number of important genetic and epigenetic mechanisms that cause alterations in growth and differentiation genes in prostate cancer. In addition to DNA deletion and point mutation, DNA methylation represents a new paradigm for the inactivation of tumor suppressor or growth suppressor genes. The identification of new genes, including a prostate cancer susceptibility locus, may furnish further insight into the molecular characteristics of prostate cancer and permit the early identification of affected individuals.

Chromosome Deletion↗
PubMed1995

E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas.

Expression of the Ca(2+)-dependent, homotypic cell:cell adhesion molecule, E-cadherin (E-cad), suppresses tumor cell invasion and metastasis in experimental tumor models. Decreased E-cad expression is common in poorly differentiated, advanced-stage carcinomas. These data implicate E-cad as an "invasion suppressor" gene. The mechanism by which E-cad is silenced in advanced stage carcinomas is unclear. In this report, we show that: (a) the 5' CpG island of E-cad is densely methylated in E-cad-negative breast and prostate carcinoma cell lines and primary breast carcinoma tissue but is unmethylated in normal breast tissue; (b) treatment with the demethylating agent, 5-aza-2'-deoxycytidine, partially restores E-cad RNA and protein levels in E-cad-negative breast and prostate carcinoma cell lines; and (c) and E-cad promoter/CAT construct is expressed in both E-cad-positive and -negative breast and prostate carcinoma cell lines, indicating that these cells have the active transcriptional machinery necessary for E-cad expression. Our data demonstrate that frequent loss of E-cad expression in human breast and prostate carcinomas results from hypermethylation of the E-cad promoter region.

Base Sequence↗
PubMed1995

Ureteroscopic endopyelotomy in an ectopic kidney.

A patient with an ectopic, pelvic kidney with congenital ureteropelvic junction obstruction was treated by retrograde, ureteroscopic endopyelotomy without percutaneous access. Preoperative ureteral stent placement and angiography facilitated the procedure. The patient had an uneventful recovery with improved drainage from the involved collecting system demonstrated by renal scintigraphy 6 months postoperatively. Ureteroscopic endopyelotomy avoids the need for percutaneous access, which may be difficult in patients with ectopic, malrotated kidneys and appears particularly well suited to the treatment of ureteropelvic junction obstruction in such cases.

Female↗