Plasma levels of IGF-1, IGF-2, and IGFBP-3 in white and African-American men at increased risk of prostate cancer.

OBJECTIVES: To further investigate the relationship between the plasma levels of insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), insulin-like growth factor binding protein-3 (IGFBP-3), growth hormone, testosterone, and demographic factors, particularly race, within a group of men at increased risk of prostate cancer development. METHODS: Enzyme-linked immunosorbent assays or an immunosorbent assay was used to quantitate the plasma levels of IGF-1, IGF-2, IGFBP-3, growth hormone, and testosterone. The study group consisted of 169 men (85 African-American, 84 white) aged 35 to 69 years, with no personal history of prostate cancer, but having at least one first-degree relative diagnosed with the disease, unless they were African-American. The relationships between the plasma levels and the categorical covariates were assessed using the nonparametric Wilcoxon test and between the continuous variables using Spearman's correlation coefficient. RESULTS: The mean plasma levels of IGFBP-3 were significantly lower in African-American (2657 ng/mL) than in white (2965 ng/mL) men (P = 0.0062). The plasma levels of IGF-2 were also lower in the African-American (503.5 ng/mL) than in the white (549.1 ng/mL) men (P = 0.0084). Overall, the IGF-1 plasma levels correlated positively with the IGF-2, IGFBP-3, and growth hormone levels and the IGF-2 plasma levels correlated negatively with the testosterone levels. CONCLUSIONS: Our results demonstrate that lower plasma levels of IGFBP-3 and IGF-2 are associated with race in a population of men at increased risk of developing prostate cancer. The ability of these markers to predict earlier disease onset is currently under investigation.

Increased hypoxia correlates with increased expression of the angiogenesis marker vascular endothelial growth factor in human prostate cancer.

OBJECTIVES: To test the hypothesis that increasing levels of hypoxia are associated with increased expression of vascular endothelial growth factor (VEGF) in prostate cancer by correlating the level of median tissue oxygenation in human prostate tumors with the immunohistochemically determined level of VEGF expression. METHODS: Custom-made Eppendorf oxygen microelectrodes were used to quantitate the pO(2) levels in prostate tumors of 13 men undergoing radical prostatectomy. All pO(2) measurements were performed under fluorine-based general anesthesia. Paraffin-embedded tumor tissue from these men was analyzed to measure the level of VEGF expression by immunohistochemical staining. The significance of the associations between the pO(2) levels and VEGF staining were determined by the Pearson correlations. RESULTS: The range of the median pO(2) levels (based on between 97 and 129 individual measurements) among 13 prostate tumors was 0.5 to 44.9 mm Hg. The blinded comparison of pO(2) levels and VEGF staining intensity demonstrated a significant correlation between increasing hypoxia and the percentage of cells staining positive for VEGF (r = -0.721, P = 0.005). This correlation was also significant when pO(2) levels were compared with the overall immunoreactive score, which takes into account staining intensity (r = -0.642, P = 0.018). CONCLUSIONS: To our knowledge, this is the first study demonstrating a significant association between increasing levels of hypoxia and increased expression of the angiogenesis marker VEGF in human prostate carcinoma. The results of our study further support the exploration of antiangiogenesis strategies for the treatment of human prostate cancer.

Loss of the short arm of the Y chromosome in human prostate carcinoma.

A change in Y chromosome number is one of the many cytogenetic abnormalities reported in human prostate tumors. However, reports in the literature have varied regarding the frequency of Y loss or gain and the significance of Y aneusomy with respect to the biology of the disease. We have conducted an analysis of the Y chromosome in malignant and benign hyperplastic human prostate epithelium in order to determine whether regional Y loss occurs in prostate cancer. To accomplish this we performed dual-color fluorescence in situ hybridization (FISH) on serial sections of paraffin-embedded prostate tumor tissues using either a Yp (SRY), Ycen (alpha-satellite) or Yq (satellite 3) probe, and an Xcen (alpha-satellite) probe that served as a control for hybridization and nuclear truncation. The results of our FISH analysis demonstrated loss of Yp in the malignant epithelium of 14/40 (35%) prostate tumor sections examined. We also found loss of Yq in 4/40 (10%) of the samples, with one of these exhibiting accompanying Yp loss. The remaining samples, 23/40 (58%), retained both Yp and Yq markers, with no evidence of either Ycen loss or Y gain in any of the tumor samples examined. In addition, Y loss was detected in the benign hyperplastic regions in nearly one-half of the tissue sections that exhibited Y loss in the malignant epithelium. These results demonstrate that regional chromosome Y loss occurs in prostate cancer, that loss of Yp is the most frequent event, and suggest that this loss may in some cases be a precursor to prostate malignancy.

CLAR1, a novel gene that exhibits enhanced expression in advanced human prostate cancer.

The molecular events involved in prostate cancer progression are, at present, poorly understood. Using a differential display technique, we identified a cDNA fragment that is present in greater abundance in stage D prostate tumors compared to stage B tumors. Northern analysis was used to confirm that transcripts for this gene are expressed at higher levels in prostate tumors of later pathological stage and higher Gleason grade compared to tumors of earlier stage and lower grade. These transcripts were also expressed at high levels in all four human prostate cancer cell lines, the neonatal prostate cell line FNC 267beta1, and in a variety of other normal human adult and fetal tissues. The cDNA fragment obtained by differential display was used as a probe to clone the full-length cDNA for this gene from a human heart cDNA library. DNA sequence analysis confirmed that the cDNA was novel, and we have named this gene CLAR1. The gene displays two transcripts of 2.6 and 2.0 kb in all tissues examined. CLAR1 maps to chromosome 19q13.3 and appears highly conserved among mammals. The deduced amino acid sequence of CLAR1 encodes a proline-rich protein that contains several SH3-binding domains and a serine phosphorylation site. The presence of these motifs suggests a possible role for CLAR1 in one or more signal transduction pathways. The enhanced expression of this novel gene in more advanced forms of prostate cancer and its potential role in signal transduction both argue that this gene should be further investigated.

Racial differences in insulin-like growth factor binding protein-3 in men at increased risk of prostate cancer.

OBJECTIVES: To determine the overall plasma levels of insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) in a group of men at higher risk of prostate cancer development and to investigate the relationships between demographics and these levels, particularly with regard to race. METHODS: An enzyme-linked immunosorbant assay was used to quantitate plasma levels of IGF-1 and IGFBP-3. The study group consisted of 105 men (63 African American [AA], 42 white), aged 35 to 69 years, with no personal history of prostate cancer, but having at least one first-degree relative diagnosed with the disease, unless they were AA. Differences in plasma levels and categorical covariates were assessed using the nonparametric Wilcoxon test. Associations between plasma levels and the continuous variables were quantified using the nonparametric Spearman correlation coefficient. RESULTS: The mean plasma level of IGF-1 was not significantly different between AA (162.3 ng/mL) and white (172.1 ng/mL) men (P = 0.415). However, the mean plasma level of IGFBP-3 was lower in AA (2789 ng/mL) than in white (3216 ng/mL) men, and this decrease was highly significant (P = 0.0045). No correlation between IGFBP-3 plasma level and age was detected in the group as a whole, but an inverse relationship between IGF-1 plasma level and age was evident (P = 0.0079). CONCLUSIONS: Our results demonstrate that IGFBP-3 plasma levels are lower in AA men than in white men. Since IGFBP-3 can control IGF-1 bioavailability, the lowered IGFBP-3 could explain in part the increased risk of prostate cancer in AA men.

Y chromosome enumeration in touch preparations from 42 prostate tumors by interphase fluorescence in situ hybridization analysis.

A change in Y chromosome number is but one of the many cytogenetic abnormalities reported in human prostate tumors. However, reports in the literature have varied regarding the frequency of Y loss or gain, whether it is restricted to the cancerous tissue, and its relation to the biology of the disease. The most frequently used materials for analysis of Y enumeration have been metaphase spreads from short-term cell cultures of prostate tumor tissue and paraffin-embedded tissue sections. Analysis of Y chromosome number by using metaphase spreads on short-term cultures can be misleading owing to clonal cell selection during the establishment of these cultures. This may result in an incomplete representation of the loss/gain pattern in the tumor as a whole. Studies using paraffin-embedded tissue sections can be complicated by apparent chromosome loss due to nuclear truncation as a result of tumor sectioning. In an attempt to circumvent these problems, we have used touch preparations from human prostate tumors to search for Y chromosome loss. Fluorescence in situ hybridization analysis was conducted by using a whole chromosome Y paint, with an alpha-satellite chromosome 3 probe as a control, on tumor samples from 42 patients ages 40-75. The results demonstrated a gain of Y in a single prostate tumor sample, with no convincing evidence for loss of the entire Y chromosome in any of the other 41 samples examined. The results suggest that loss of the entire Y chromosome is an infrequent event in prostate cancer.

Prostate cancer risk assessment program. A model for the early detection of prostate cancer.

Prostate cancer is the most common form of cancer (except skin cancer) in men. Several factors have been associated with an increased risk for prostate cancer, including age, ethnicity, family history, lifestyle, and environmental exposures. Recognition of the importance of the interaction of these factors in prostate cancer has led to an interest in their evaluation as a model both for studying genetic susceptibility patterns and for studying and providing educational tools and preventive interventions. One such model has been developed at Fox Chase Cancer Center. Critical to the implementation of the model has been the establishment of the Prostate Cancer Risk Registry (PCRR) and Prostate Cancer Risk Assessment Program (PRAP). Together, they serve as a unique resource for investigating the interaction between environmental factors and genetic susceptibility patterns; exploring the early, premalignant biological markers of prostate cancer; and prospectively assessing the quality of life (QOL) of men at risk. In addition, PRAP facilitates the evaluation of models for prostate cancer risk counseling and screening in the community. This paper describes this model for early detection and risk reduction, along with preliminary data from its first two study aims. The program is particularly relevant in view of the wealth of genetic information emerging from the Human Genome Project.

Overexpression of cyclin D1 is rare in human prostate carcinoma.

BACKGROUND: Overexpression of cyclin D1 has been documented in a number of human cancers. Increased expression of cyclin D1 can contribute to cellular transformation and abnormal proliferation. METHODS: Quantitative RT-PCR and/or Western blot analysis were used to determine the level of cyclin D1 expression in 96 human prostate tumors, 15 benign prostate hyperplasias, 4 prostate cancer cell lines, and 3 xenografts. RESULTS: Our results demonstrate that 4.2% of the prostate tumors examined overexpressed cyclin D1 transcripts. In the cell lines, expression was normal, with the exception that reduced levels of cyclin D1 transcript and protein were observed in the DU145 cell line, as expected from cells with mutant RB. Normal levels of cyclin D1 were found in all xenograft tumors and BPH specimens examined. CONCLUSIONS: These data show that overexpression of cyclin D1 occurs rarely in human prostate tumors. However, when overexpression of cyclin D1 does occur, it may identify a subset of tumors with a different molecular biology.

Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in late pathological stage human prostate cancer.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme best known to many investigators as a 'housekeeping' gene used as a loading control on northern blots. Prior studies, however, have shown that GAPDH RNA levels vary greatly among different prostate cancer cell lines. We undertook this study to determine the level of GAPDH gene expression within primary human prostate tumors and to determine the validity of using GAPDH as a loading control for northern analysis of human prostate cancer specimens and normal human organs. We found that GAPDH expression was significantly higher in specimens from patients with pathological stage C and D prostate cancer than those with stage B disease. Within the human prostate cancer cell lines TSUPr1, DU145, LNCaP and PC-3 and a normal neonatal prostate epithelial cell line FNC 267beta1, LNCaP cells expressed the highest level of GAPDH but all cell lines, including the normal neonate prostate cell line had very strong GAPDH gene expression. GAPDH RNA levels were highly variable among 16 normal human adult organs and four human fetal organs examined. We conclude that GAPDH RNA levels in human prostate tumors correlate with pathologic stage and that GAPDH should not be used as a loading control in northern blot experiments. Other controls, such as beta-actin or 28s ribosomal RNA, would be more suitable for such purposes.

Alterations of the retinoblastoma gene in human prostate adenocarcinoma.

The loss or mutational inactivation of the RB1 tumor suppressor gene has been implicated in the development of a diverse group of human malignancies. However, the contribution of the RB1 gene alteration to human prostatic carcinogenesis has been poorly understood. Thus far, deletion of the promoter sequence and exon 21 from one primary tumor specimen and the alterations found in the cell line DU-145, are the only cases of RB1 mutations reported in human carcinoma of the prostate. This study was designed to determine whether alterations in the structure or expression of the RB1 gene occur in human prostate carcinoma, and to determine the nature of these changes and the frequency with which they occur. One hundred twelve primary prostate tumor tissues and four metastatic lesions were obtained immediately after surgical resection. The RB1 gene was characterized in 68 tumor DNA samples using Southern analysis and the PG3.8M or H3-8 probes. Band profiles were analyzed by scanning densitometry. Sixty-three tumor DNA samples were analyzed for defects in the RB1 promoter using polymerase chain reaction (PCR) and heteroduplex analysis. Alterations in the expression of exons 1-27 were analyzed in 79 primary and four metastatic tumor RNAs using RT-PCR. Three of 68 tumors were identified to have gross rearrangement of the RB1 gene or deletion of one allele. One of four stage D tumor specimens showed truncated RT-PCR products indicating an internal deletion of RB1 transcripts. In all, 14 of 83 (17%) specimens displayed abnormally low levels of RB1 mRNA expression. Furthermore, these alterations of RB1 expression showed a correlation with increasing tumor stage and grade. These results suggest alterations of RB1 mRNA expression occur more frequently in higher stages and grades of prostate cancer and, thus, may be contributing to the malignant progression of a subset of human prostate cancer.

Detection of sex-region Y (SRY) transcripts in human prostate adenocarcinoma and benign prostatic hypertrophy.

The human sex-region Y (SRY) gene maps to Yp11.3 and encodes a protein that shares significant sequence homology with a conserved DNA binding motif found in the nonhistone high-mobility group (HMG) proteins. In the mouse, Sry is required for normal testicular development and is expressed in the developing male gonadal ridge as well as in the adult testis. In man, SRY expression has been observed in the adult testis, but not in other adult male tissues. We have analyzed samples from human prostate adenocarcinoma and benign prostatic hypertrophy (BPH) for the expression of the SRY gene. We found expression of SRY in 60% of malignant prostate tumors and in three of six samples of BPH. We did not find expression in male or female colon mucosa, or in tissue from a cystic ovary. Malignant and atrophic testicular tissue both contained SRY transcript and served as positive controls in these experiments. We also found SRY transcript in the DU-145 prostate adenocarcinoma cell line. Interestingly, SRY expression is absent in the Tera-2 teratocarcinoma cell line. The potential for the SRY gene product to bind HMG core response elements in vitro suggests that SRY could participate in the cascade of gene regulatory events that result in aberrant cell growth or malignancy.

ZFY gene expression and retention in human prostate adenocarcinoma.

The terminal portion of the short arm of the human Y (Yp) chromosome encodes a zinc-finger DNA binding protein (ZFY). A highly homologous gene, ZFX, is encoded on Xp. The potential of the zinc finger motif for regulating the expression of other genes suggests a role for this protein in the development of malignancy. Prostate adenocarcinoma is a malignancy of male-specific tissue, the incidence of which increases beyond the fifth decade of life. We have analyzed samples of human prostate adenocarcinoma for the expression of ZFY and ZFX transcripts. We found expression of ZFY transcripts in 3 of 31 prostate adenocarcinomas by using Northern analysis. No ZFY or ZFX transcripts were detected in normal hypertrophic prostate tissue on Northern analysis. In one prostate adenocarcinoma, high levels of the 5.1 kb ZFY and the 8.0 and 6.3 kb ZFX transcripts were present. In addition, this high-grade tumor contained a novel 4.3 kb transcript. When we used reverse transcriptase PCR (RT-PCR) to analyze these same samples, the number of tumors expressing ZFY and/or ZFX transcripts increased to 20 of 31. Transcripts for these genes were also present in the DU-145 and LNCaP human prostate adenocarcinoma cell lines. In 2 of the 6 benign prostatic hypertrophy (BPH) tissues analyzed by RT-PCR, barely detectable products of ZFY were observed, and none contained ZFX products. Southern analysis revealed that the portion of the Y chromosome which contains the ZFY gene was not lost from the majority of the tumor cells in any of the prostate malignancies examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced levels of mRNA for myofibrillar proteins in skeletal muscle from septic rats.

The influence of sepsis on transcription of myofibrillar proteins in skeletal muscle was studied in rats. Sepsis was induced by cecal ligation and puncture (CLP); control rats were sham-operated. Sixteen hours later, muscle levels of mRNA for myofibrillar proteins were determined by using cDNA probes specific for transcripts for alpha actin and myosin heavy chain. Sepsis resulted in a 2-6 fold decrease in alpha actin mRNA levels and an even more pronounced reduction in myosin heavy chain mRNA levels. Results suggest that sepsis-induced reduction of muscle protein synthesis is at least partly regulated at the transcriptional level.

Western blot analysis of porcine corpus luteum heparin binding proteins using antibodies to acidic and basic fibroblast growth factor.

The 105,000 X g supernatant from homogenates of porcine corpora lutea was chromatographed on a heparin Sepharose affinity column. Bound protein was batch eluted and analyzed on Western blots using antibodies to acidic and basic fibroblast growth factor (FGF). The antibody to acidic FGF reacted specifically with at least four protein bands ranging from 21 to 50 kDa. Three antibodies to human basic FGF (145 residues) generated against either the N-terminal sequence (1-12), the internal sequence (33-43), or the C-terminal end (135-145) also reacted specifically with a total of four different bands. The apparent molecular weights ranged between 20 and 55 kDa. The luteal extract also expressed message for acidic FGF. The results show that there may exist a family of FGF-like molecules in the corpus luteum and demonstrates for the first time the presence of acidic FGF in that tissue.

Enhanced levels of insulin-like growth factor messenger RNA in human colon carcinomas and liposarcomas.

The insulin-like growth factors I and II (IGF-I and -II) are proteins which stimulate cell proliferation and are important in normal human growth and development. They are coded for by separate genes and bind to specific cell surface receptors, eliciting a mitogenic response. IGFs are secreted by several cell lines derived from adult tumors. We have examined a number of human adult tumors for IGF messenger RNA (mRNA) expression and found IGF-II mRNA levels were consistently elevated in two types, colon carcinoma and liposarcoma. Adult colonic mucosa contains low levels of IGF-I and -II mRNA while several colon tumors, particularly of rectal and rectosigmoid origin, contained significantly elevated levels of IGF-II message. Over 90% of liposarcomas examined contained greatly elevated levels of IGF-II mRNA while control tissue (adipose) contained very low or undetectable IGF mRNA levels. Many of these tumors also contained elevated IGF-I mRNA levels. Northern analysis of these RNAs revealed differences in the abundance and sizes of IGF transcripts compared to other normal and malignant tissues known to express IGF.

The gene for human transforming growth factor alpha is on the short arm of chromosome 2.

Transforming growth factor alpha is a polypeptide growth factor that participates in the reversible transformation of cells in vitro and is secreted by many transformed cell lines. It also shares sequence and functional homologies with epidermal growth factor. Working with a cloned cDNA probe (lambda hTGF1-10) and derivatives, we have mapped this gene (TGFA) to 2p13 with the use of somatic cell hybrids and in situ hybridization. This is the same region involved in the 2;8 translocations of Burkitt lymphoma. Such a rearrangement could orient c-myc (8q24) adjacent to TGFA, resulting in activation of one or both of these genes.

Assignment of the human fibronectin structural gene to chromosome 2.

A cloned human cDNA probe for fibronectin (FN) containing 1.3 kb of the human FN coding region has been used to determine the chromosome that encodes the structural gene in human-mouse somatic cell hybrids. The results show that human chromosome 2 encodes the FN structural gene.

Isolation of polymorphic DNA segments from human chromosome 21.

A somatic cell hybrid line containing only human chromosome 21 on a mouse background has been used as the source of DNA for construction of a recombinant phage library. Individual phages containing human inserts have been identified. Repeat-free human DNA subclones have been prepared and used to screen for restriction fragment length polymorphisms to provide genetic markers on chromosome 21. Nine independently isolated clones used as probes identified a total of 11 new RFLPs. Four of the DNA probes recovered from the library have been mapped unequivocally to chromosome 21 using a panel of somatic cell hybrid lines. A fifth probe detected an RFLP on chromosome 21 as well as sequences on other chromosomes. This set of RFLPs may now form the basis for construction of a genetic linkage map of human chromosome 21.

DNA topoisomerase I and II activities during cell proliferation and the cell cycle in cultured mouse embryo fibroblast (C3H 10T1/2) cells.

We have used C3H 10T1/2 cells to examine the regulation of topoisomerase activities during cell proliferation and the cell cycle. The specific activity of topoisomerase I was about 4-fold greater in proliferating (log phase) cells than in non-proliferating (confluent) cells. In synchronized cells, the bulk of the increased activity occurred during or just prior to S phase, depending upon the method of synchronization. A smaller increase in activity also occurred during G1 phase. The increase in activity during S phase was not altered by a hydroxyurea block at the G1/S phase boundary indicating that it is not directly coupled to DNA synthesis and is not the result of topoisomerase I gene dosage. The increase was inhibited by blocking cells at mid-G1 phase using isoleucine deprivation. Thus, the increase in activity during S phase is dependent on events occurring during mid- to late G1 phase. In contrast to the changes in topoisomerase I levels, the specific activity of topoisomerase II showed no detectable difference in proliferating vs non-proliferating cells. In addition, no detectable difference in topoisomerase II specific activity was seen in G1, S and M phases of the cell cycle. The differences in the activity profiles of the topoisomerases I and II during the cell cycle suggest that the two activities are regulated independently and may be required for different functions.

Localization of insulin-like growth factor genes to human chromosomes 11 and 12.

The insulin-like growth factors IGF-I and IGF-II are required for growth and development. Both are single-chain proteins (of 70 and 67 amino acids respectively) derived from precursors by proteolytic processing. IGF-I may be particularly important in promoting normal stature and IGF-II may be a fetal growth hormone. The IGF proteins are probably synthesized by many normal tissues and by some tumours. The secretion of growth factors by tumours and tumour-derived cell lines suggests that they may act as autocrine regulators of cell proliferation. Because of the possible role of these proteins in growth disorders and cancer, and their sequence homology with insulin, we have determined their chromosomal localization. Using somatic cell hybrids and cloned cDNA probes for these proteins, we have assigned the genes for IGF-I and IGF-II to human chromosomes 12 and 11, respectively. We present evidence that the IGF-II gene is located on the short arm of chromosome 11 with a ras proto-oncogene and the insulin structural gene, and also suggest the existence of a fragment length polymorphism using the IGF-I probe.