INSECT PHYLOGENOMICS. Response to Comment on "Phylogenomics resolves the timing and pattern of insect evolution".

Tong et al. comment on the accuracy of the dating analysis presented in our work on the phylogeny of insects and provide a reanalysis of our data. They replace log-normal priors with uniform priors and add a "roachoid" fossil as a calibration point. Although the reanalysis provides an interesting alternative viewpoint, we maintain that our choices were appropriate.

Tumor-suppression function of transcription factor USF2 in prostate carcinogenesis.

Although the transcription factor USF2 has been implicated in the regulation of cellular growth and proliferation, it is unknown whether alterations in USF2 contribute to tumorigenesis and tumor development. We examined the role of USF2 in prostate tumorigenesis. Western blot analysis revealed markedly decreased USF2 levels in three androgen-independent prostate cancer cell lines, PC-3, DU145, and M12, as compared to nontumorigenic prostate epithelial cells or the androgen-dependent cell line, LNCaP. Ectopic expression of USF2 in PC-3 cells did not affect the cell proliferation rate of PC-3 cells on plastic surfaces. However, it dramatically decreased anchorage-independent growth of PC-3 cells in soft agar (90-98% inhibition) and the invasion capability (80% inhibition) of PC-3 cells in matrix gel assay. Importantly, expression of USF2 in PC-3 cells inhibited the tumorigenicity of PC-3 cells in an in vivo nude mice xenograft model (80-90% inhibition). These results suggest that USF2 has tumor-suppression function. Consistent with its function in tumor suppression, we found that the USF2 protein is present in normal prostate epithelial cells but absent in 18 of 42 (43%) human prostate cancer tissues (P = 0.015). To further examine the functional role of USF2 in vivo, we generated mice with genetic deletion of USF2 gene. We found that USF2-null mice displayed marked prostate hyperplasia at a young age, suggesting that USF2 is involved in the normal growth and differentiation of prostate. Together, these studies demonstrate that USF2 has tumor-suppressor function and plays a role in prostate carcinogenesis.

Regulation of proliferation of prostate epithelial cells by 1,25-dihydroxyvitamin D3 is accompanied by an increase in insulin-like growth factor binding protein-3.

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to regulate the proliferation of human prostate epithelial cell lines. Since the insulin-like growth factor (IGF) system is involved in the transformation process of epithelial cells, the following study was undertaken to determine if the IGF system, in particular IGF binding protein-3 (IGFBP-3), is altered by 1,25-(OH)2D3 in normal prostate epithelial cells as part of a mechanism for inhibition of transformation. Two cell systems were used in this study: (1) primary cultures of benign human prostate epithelial cells (PECs) and (2) an SV40-T immortalized prostate epithelial cell line (P153) that is non-tumorigenic. 1,25-(OH)2D3 was added to parallel sets of PECs and P153 cells in addition to the presence or absence of IGF-I or des(1-3)IGF-I. Treatment with 1,25-(OH)2D3 resulted in significant growth inhibition of both PECs and P153 cells. Furthermore, 1,25-(OH)2D3 inhibited IGF-induced proliferation, but this was partially reversed by high concentrations of IGF-I. Western ligand blots of condition media demonstrated a significant increase in IGFBP-3; likewise Northern blots demonstrated an increase in mRNA for IGFBP-3. Proliferation assays using an antibody designed to block the IGF-independent effects of IGFBP-3 failed to reverse the inhibitory effect of 1,25-(OH)2D3. Thus, IGFBP-3 acts in an IGF-dependent manner to inhibit cell growth of benign prostate epithelial cells.

A novel mechanism for chaperone-mediated telomerase regulation during prostate cancer progression.

Telomerase activity has been detected in >85% of all malignant human cancers, including 90% of prostate carcinomas. Using a well-characterized experimental prostate cancer system, we have found that telomerase activity is notably increased (>10-fold) during tumorigenic conversion. Expression profiles of the telomerase components (hTR and hTERT) revealed no substantive changes, which suggests a nontranscriptional mechanism for increased activity. Because the hsp90 chaperone complex functionally associates with telomerase, we investigated that relationship and found that along with telomerase activity, a number of hsp90-related chaperones are markedly elevated during transformation, as well as in advanced prostate carcinomas. Using the nontumorigenic cell protein extract as the source of telomerase, addition of purified chaperone components enhanced reconstitution of telomerase activity, which suggests a novel mechanism of increased telomerase assembly via a hsp90 chaperoning process during prostate cancer progression.

Suppression of tumorigenicity in the human prostate cancer cell line M12 via microcell-mediated restoration of chromosome 19.

Previously we immortalized human, nontransformed prostate epithelial cells with SV40 large T-antigen (SV40TAg) and derived increasingly aggressive sublines from the immortalized line. The progression of the tumorigenic sublines to metastatic capacity was accompanied by the formation of an unbalanced translocation between chromosomes 16 and 19, resulting in loss of 19p and proximal 19q. To test whether the tumorigenic and/or metastatic phenotype was causally related to this genetic alteration, we restored a neo-tagged human chromosome 19 to M12 cells by microcell-mediated transfer and assessed their growth. In vitro, the resultant hybrids grew more slowly in monolayer culture and showed a significant reduction in anchorage-independent growth as compared to M12neo controls. In vivo, all mice (13/13) injected subcutaneously (SC) with control M12neo cells developed tumors after 9-15 days. In contrast, 9/15 mice injected SC with microcell-transferred chromosome 19 hybrid cells failed to form tumors, with 6/15 producing very small tumors after 120 days. Analysis of three of these six tumors showed consistent, new chromosomal changes. Furthermore, in one of the tumors, loss of a chromosome 19 was noted in 40% of the cells. After intraprostatic injections of the hybrid cells, only 2/7 mice developed microscopic tumors, with no metastases. These data suggest the presence of a gene or genes on chromosome 19 that function to suppress growth.

Transcriptional regulation of insulin-like growth factor-I receptor gene expression in prostate cancer cells.

A marked decrease in the type 1 insulin-like growth factor (IGF) receptor (IGF-IR) occurs in prostate epithelial cells during transformation from the benign to the metastatic state. One of the principal regulators of IGF-IR gene expression, the WT1 tumor suppressor, is expressed in prostate cancer and in prostate cancer cell lines. The purpose of this study was to determine whether the decrease in IGF-IR expression was transcriptionally regulated, and whether WT1 action may be involved in the repression of the IGF-IR gene in prostate cancer cells. The P69 cell line was derived by immortalization of human primary prostate epithelial cells with simian virus-40 T antigen and is rarely tumorigenic. The M12 line was derived from the P69 line by selection for tumor formation in nude mice and is tumorigeneic and metastatic. P69 cells express 20,000 IGF-IR/cell, whereas M12 cells express 3,500 IGF-IR/cell. These differences in receptor number are reflected in proportional differences in IGF-IR mRNA levels. To assess IGF-IR promoter activity in these cell lines, each was transiently transfected with luciferase reporter vectors containing the IGF-IR gene transcription start site and 476 bp of 5'-flanking sequence, 640 bp of 5'-untranslated region sequence, or both regions. The promoter activity of the full-length construct was 50% lower (P < 0.01) in M12 cells compared with P69 cells, the activity of the 5'-flanking region construct was 53% lower (P < 0.0001), and that of the 5'-untranslated region construct was 36% lower (P = 0.01). P69 clones stably transfected with a WT1 expression vector exhibited decreased expression of the endogenous IGF-IR gene and decreased promoter activity in transient transfection assays with IGF-IR promoter constructs containing multiple WT1 binding sites. The observed reduction in endogenous IGF-IR expression was sufficient to inhibit IGF-I-stimulated cell proliferation. These data suggest that most of the decreased expression of the IGF-IR seen in malignant prostate epithelium is the result of transcriptional repression of the IGF-IR gene, and that this repression may be due in part to the increased expression of the WT1 tumor suppressor in metastatic prostate cancer.

Detection of a novel truncated WT1 transcript in human neoplasia.

BACKGROUND: The Wilms' tumor 1 (WT1) gene encodes a transcription factor critical in urogenital development. Using a new model of prostate cancer progression that permits comparison of the cellular and molecular properties of increasingly aggressive sublines of simian virus 40 large T-antigen-immortalized human prostate epithelial cells within the same lineage, the role of WT1 in tumorigenesis was investigated. METHODS AND RESULTS: Using RT-PCR and northern blotting, we identified a novel truncated WT1 transcript in these prostate cancer cell lines. This 2.1-kb transcript consisted of the coding region of the zinc-finger domain of WT1, together with a portion of intron 5 at the 5' end of the transcript. Furthermore, two peptides were detected by western blotting using antibodies to epitopes of the COOH terminus of WT1. Using RT-PCR, the 2.1-kb transcript was also detected in leukemia cell line K562, breast cancer cell line MCF7, and blood samples from patients with acute leukemia. CONCLUSION: These novel findings in both cell lines and patient-derived specimens suggest this new WT1 gene alteration has a potential role in the development of new diagnostic assays for some human malignancies.

Implementation of automation in a small-scale DNA sequencing core facility.

New England Biolabs (NEB) sequencing core facility provides automated sequencing services to support various company-wide projects in house, but on a very small scale of about 1000 to 1500 reactions per month. A procedure has been implemented at the NEB core sequencing facility to integrate simplified methods and robotics to provide a more efficient small-scale process. This has been done using a Beckman Biomek 2000 robot combined with an MJ DNA Engine, 96-well plate cycler (PTC-200), AB 373 and 377 sequencers, BMA Singel gels, and several other materials that help reduce the time required for otherwise lengthy procedures in a cost-efficient manner. Protocols have also been developed for efficient sequencing of a variety of templates submitted to the NEB core facility.

Overexpression of an inhibitory insulin-like growth factor binding protein (IGFBP), IGFBP-4, delays onset of prostate tumor formation.

Insulin-like growth factor (IGF) binding proteins (IGFBPs) have been shown to either inhibit or enhance the action of IGF, or act in an IGF-independent manner in the prostate. We have overexpressed the IGF-inhibitory IGFBP-4 in the malignant M12 prostate epithelial cell line to determine the effects on tumor formation and apoptosis. Overexpression was determined by Northern, Western immunoblot and Western radioligand blot analysis. IGF-induced proliferation was reduced in the IGFBP-4 transfected cells compared with control cells (P < or = 0.01). Colony formation in soft agar was significantly inhibited up to 14 days after plating in the IGFBP-4 transfected cells when compared with the M12 controls (P < or = 0.01): however, in the presence of des(1-3)IGF-I, there was no significant difference between the control and IGFBP-4 transfectants in colony formation in soft agar. Apoptosis in an IGFBP-4 transfected cell line was significantly increased in response to induction by 6-hydroxyurea compared with the control line. When injected s.c. into male athymic/nude mice, a marked delay was noted in tumor formation in animals receiving IGFBP-4 transfected cells (P < or = 0.01). Interestingly, IGFBP-2 protein levels were reduced in the conditioned media of all IGFBP-4 transfected cell cultures. These data indicate that an inhibitory IGFBP may significantly delay the growth of malignant prostate epithelial cells and enhance the sensitivity of these cells to apoptosis.

Metastatic sublines of an SV40 large T antigen immortalized human prostate epithelial cell line.

BACKGROUND: The available human prostate cancer cell lines that are metastatic in athymic nude mice all have complex, highly aneuploid karyotypes. Other prostatic cells immortalized by transforming genes of SV40 or HPV and converted to tumorigenicity by additional genetic manipulation are not reported to be metastatic. METHODS: Tumorigenic sublines of human prostate epithelial cells previously immortalized by transfection with the SV40T antigen gene were obtained by sequential passage in male athymic nude mice. These sublines were evaluated histopathologically for tumorigenicity and metastasis in athymic nude mice after subcutaneous, intraperitoneal, and intraprostatic injection. Each subline was characterized by standard (GTG-banding) cytogenetic and FISH analysis, and RNase protection assays for androgen receptor expression. RESULTS: Two sublines produced metastases in lungs and the diaphragm of most mice after either intraprostatic or intraperitoneal injection. The M2205 subline formed large local tumors after intraprostatic injection. Cytogenetic aberrations present in the metastatic sublines, but not in the tumorigenic, nonmetastatic lines or the parental P69SV40T line, included dup(11)(q14q22), der(16) t (16;19) (q24;q13.1), which resulted in the loss of the short arm and proximal long arm of chromosome 19 (19q13.1-->19pter), and loss of the Y chromosome. None of the sublines expressed the androgen receptor. CONCLUSIONS: These cytogenetically defined, SV40T-immortalized human prostate epithelial cell lines, with distinct biological behaviors in vivo, provide additional tools for the genetic analysis of the emergence of metastatic capacity.

Hevin, an antiadhesive extracellular matrix protein, is down-regulated in metastatic prostate adenocarcinoma.

Hevin, a gene closely related to the extracellular matrix protein SPARC, is an acidic cysteine-rich glycoprotein shown to be important for the adhesion and trafficking of cells through the endothelium. Through the use of differential display and differential EST analysis, we identified Hevin as a gene whose transcription is down-regulated in transformed prostate epithelial cell lines and metastatic prostate adenocarcinoma. These results were confirmed by comparing expression levels between normal and neoplastic human prostate tissues using Northern analysis. In situ hybridization with an 35S-labeled antisense riboprobe demonstrated the loss of Hevin expression in metastatic prostate carcinoma. The expression pattern of Hevin in transformed and metastatic epithelium may provide further insights into the complex cell adhesion events involved in the metastatic progression of prostate carcinoma.

Growth factor network disruption in prostate cancer progression.

Prostate cancer progression is often accompanied by alterations in expression of one or more growth factor receptors and/or their ligands. This commentary examines the hypothesis that the disruption of the normal growth factor network itself is a critical event in prostate cancer progression. If such transition points are proven, there are significant ramifications.

Type-1 insulin-like growth factor receptor reexpression in the malignant phenotype of SV40-T-immortalized human prostate epithelial cells enhances apoptosis.

The authors have previously shown that the type 1 insulin-like growth factor receptor (IGF-1R) is decreased in the transformation from benign to malignant human prostate epithelial cells in vivo. Further, in a well-described human SV40-T immortalized human epithelial cell system beginning with the immortalized, but rarely tumorigenic P69SV40-T cell line, to the highly tumorigenic and metastatic M12 subline, there is a similar decrease in IGF-1R number from 2.0 x 10(4) receptors per cell to 1.1 x 10(3) receptors per cell. When the IGF-1R was reexpressed in the M12 subline using a retroviral expression vector, M12-LISN, to a receptor number similar to that of the P69SV40-T parental cell line, the authors demonstrated a marked decrease in colony formation in soft agar in the M12-LISN cells vs the M12 control cells (p < or = 0.01), and a decrease in vivo tumor growth and metastases when injected either subcutaneously or an intraprostatic location (p < or = 0.01). This decrease in tumor volume was not because of a decrease in proliferative capacity, but was associated with an increase in apoptosis in baseline cultures and in response to the apoptotic-inducing agents 6-hydroxyurea, retinoic acid, and transforming growth factor beta 1.

Economic advantages of breast-feeding in an HMO: setting a pilot study.

We performed a pilot study on newborns randomly chosen from term singleton deliveries born to mothers in an HMO group between September 1992 and August 1993. Breast-fed infants were breast-feeding at 6 months (n = 41), whereas bottle-fed infants were bottle-fed from birth (n = 107), Medical care and costs for the first 12 months were retrospectively analyzed, including office visits, drug prescriptions, and hospitalizations. Both groups had similar numbers of office visits and pharmacy costs. Breast-fed infants had fewer inpatient admissions (0.13 vs. 0.20 discharges per 1,000 babies), and their average total medical costs were $200 less than those of bottle-fed infants. Extrapolating to the total number of deliveries during this period, an increase in breast-feeding from the current rate (17%) to the Healthy People 2000 goal (50%) could save up to $140,000 annually.

Reexpression of the type 1 insulin-like growth factor receptor inhibits the malignant phenotype of simian virus 40 T antigen immortalized human prostate epithelial cells.

Type 1 insulin-like growth factor receptor (IGF-1R) expression is decreased in prostate cancer compared to that in noncancerous prostate epithelium. We have demonstrated that as the simian virus 40 T antigen (SV40T) immortalized human prostate epithelial cell line, P69SV40T, undergoes transformation from a poorly tumorigenic to a malignant phenotype, the M12 subline, there is a significant decrease in IGF-1R expression. In the present study, we examine the effects of reexpression of the IGF-1R on the malignant phenotype of M12 cells. The IGF-1R was reexpressed in M12 cells using a retroviral vector containing a 7-kilobase coding sequence for the IGF-1R, LISN, to create several clones of the M12-LISN cell line. As a control, M12 cells were also infected with a retroviral vector (LNL6) without the 7-kilobase IGF-1R insert (M12-LNL6 clones). Functional assays were performed with two separate clones each of M12-LNL6 and M12-LISN cells. Each clone of M12-LISN cells regained the proliferative response to IGF that was lost in the transition from P69SV40T cells to M12 cells. In addition, M12-LISN clones had a significantly decreased growth rate compared to the M12-LNL6 cells when injected s.c. in athymic/nude mice (P < 0.001). Tumorigenicity, as assessed by anchorage-independent growth of colonies in soft agar, was also decreased by 75% in the M12-LISN clones compared to that in the M12-LNL6 control cells. These data demonstrate that reexpression of the IGF-1R in a malignant human prostate epithelial cell line results in decreased tumor growth and decreased anchorage-independent colony formation independent of an increased proliferative response to IGF. Reexpression of the IGF-1R may be associated with reacquisition of the regulation of cellular proliferative and differentiation functions mediated by the IGF-1R that are lost as prostate epithelial cells undergo conversion to a malignant phenotype.

The effect on the insulin-like growth factor system in human prostate epithelial cells of immortalization and transformation by simian virus-40 T antigen.

The insulin-like growth factor (IGF) system has been demonstrated to be important for proliferation and differentiation in tissues. This system has also been demonstrated to be an important regulator of the growth of normal prostate epithelium and has been implicated in the process of transformation to human epithelial prostate cancer. This study examined the function of the various components of the IGF system in benign prostate epithelium (BPE), simian virus-40 (SV40)-T antigen-immortalized prostate epithelial cells, P69SV40-T (P69), and two sublines generated from the parental line by serial passage through athymic mice: one tumorigenic (M2182) and one metastatic (M12). IGF-II messenger ribonucleic acid (mRNA) and protein were detected in BPE cells, and each of the three P69 cell lines. IGF-II protein levels were significantly higher in medium collected from the P69, M2182, and M12 cells than in BPE. Proliferation in response to IGF was P69 > BPE > M2182 > M12. The proliferative responses in the four cell types were paralleled by an increase in c-jun. In addition, as the cells became progressively more tumorigenic, the basal level of c-jun mRNA increased. IGF-binding protein-2 (IGFBP-2), -3, -4, -5, and -6 could be detected in the primary epithelial cell medium; however, as the cells became progressively more tumorigenic, there was a decrease in IGFBP-2, -3, -5, and -6 in the medium. The type 1 IGF receptor (IGFr) also decreased as the cells became more tumorigenic. The M12 cells had 80% fewer receptors than the P69 cells and 70% fewer than M2182 cells. There was no change in the Kd for IGF between the cell lines. Based on these data it would appear that the difference in proliferation between the BPE cells and P69s may be due to an increased concentration of inhibitory IGFBPs in the P69 medium. The decrease in proliferation seen in response to IGF in M2182 and M12 cells compared to the P69s would appear at least in part to be due to a decreased IGFr number. IGFr mRNA is represented by 11.0- and 7.0-kilobase bands in the BPE and P69 cells, but only by an 11.0-kilobase band in M2182 and M12 cells. These data indicate that there are significant changes that occur in the IGF system during the process of malignant transformation of the prostate epithelium. The changes described in the P69 cell system are similar to those seen in vivo and suggest that an intact IGF system may be important in maintaining a differentiated epithelial cell.

Cathepsin D and epidermal growth factor receptor immunohistochemistry does not predict recurrence of prostate cancer in patients undergoing radical prostatectomy.

PURPOSE: We determined if immunohistochemical expression of the epidermal growth factor receptor and cathepsin D in the primary tumor was of prognostic value in clinically localized prostate cancer after radical prostatectomy. MATERIALS AND METHODS: Immunohistochemical staining for epidermal growth factor receptor and cathepsin D was performed on 105 radical prostatectomy specimens from 2 academic centers. The epidermal growth factor receptor and cathepsin D expressions were graded using H scoring by an experienced pathologist blinded to other patient data, and compared with age, grade, stage, race and initial serologic (prostate specific antigen) recurrence. Univariate and multivariate statistical testing was performed. RESULTS: Immunohistochemically detectable epidermal growth factor receptor and cathepsin D expression was not correlated to age, race, stage or Gleason grade. In univariate and multivariate testing epidermal growth factor receptor and cathepsin D were not prognostic markers for disease progression following radical prostatectomy. CONCLUSIONS: Immunohistochemical analysis of the biomarkers cathepsin D and epidermal growth factor receptor in radical prostatectomy specimens does not predict disease recurrence. Further biological marker study is needed in clinically localized prostate cancer.

Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity.

A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus stearothermophilus N3468 was prepared to near-homogeneity. The dominant species of the Bst DNA polymerase I preparation sized about 97 kDa when analyzed on SDS polyacrylamide gels. The Bst polA gene that codes for Bst polymerase I was cloned and sequenced. Comparative sequence analysis showed that all three conserved 3'-->5' exonuclease motifs found in E. coli DNA polymerase I were missing in Bst DNA polymerase I. This cast doubt on the existence of a 3'-->5' exonuclease function in that enzyme. Four biochemical assays were used to measure exonuclease activities of Bst DNA polymerase I, testing both full-length Bst polymerase I and the Bst large fragment which lacks the N-terminal 5'-->3' exonuclease domain. These exonuclease assays demonstrated that Bst DNA polymerase I only contained a double-strand dependent 5'-->3' exonuclease activity but lacked any detectable 3'-->5' proofreading exonuclease activity. The lack of 3'-->5' exonuclease function in a variety of thermostable repair DNA polymerases may reflect enhancement of thermostability at the expense of proofreading activity.

Proteolysis of insulin-like growth factor-binding protein-3 in the male reproductive tract.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is produced by prostate epithelial and stromal cells and either enhances or inhibits the effects of IGF on prostate epithelial cells. The levels of this protein in the male reproductive tract may be determined in part by proteases, including prostate-specific antigen (PSA), produced by the prostate epithelium. In this study we examined the proteolytic activity of human seminal fluid on IGFBP-3. Seminal fluid and prostate massage fluid (PF) were examined for IGFBP-3 or its fragments by use of an IGFBP-3 RIA that detects intact IGFBP-3 as well as fragments, a two-site immunoradiometric assay (IRMA) that detects intact IGFBP-3 and the larger fragments, Western ligand blots (WLB), and immunoblots (WIB). In seminal fluid, IGFBP-3 was readily detectable by RIA, but was detected in only 50% of the samples assayed by IRMA. No detectable IGFBP-3 was observed by WLB with [125I]IGF-I as the ligand, but with IGF-II as the ligand, IGFBP-3 fragments at 16-17 kDa were noted. On WIB, the 16-kDa fragment of IGFBP-3 was most abundant, with a smaller amount of the 29-kDa fragment, but no intact IGFBP-3. These results indicated that most of the IGFBP-3 detected in seminal fluid was in small (< or = 16-kDa) fragments. When three or more seminal fluid samples collected 1 month apart were available from the same individual, the coefficient of variation was 10.0 +/- 1.26% (+/- SE) for IGFBP-3 by RIA vs. 73.3 +/- 11.2% for sperm counts in the same samples. In a group of 42 PF samples, the IGFBP-3 levels measured by either RIA or IRMA were approximately 3-fold higher than those in seminal fluid. Intact IGFBP-3 was detected by both WLB and WIB. There was a significant inverse correlation between PSA and IGFBP-3, measured by IRMA, in PF (r = -0.526; P < or = 0.004). Finally, in the PF of African-American men, PSA was significantly lower, and IGFBP-3 determined by IRMA was significantly higher compared to those in Caucasian men.