Harry W. Carter

eIF4E Activation Is Commonly Elevated in Advanced Human Prostate Cancers and Significantly Related to Reduced Patient Survival

Elevated eukaryotic translation initiation factor 4E (eIF4E) function induces malignancy in experimental models by selectively enhancing translation of key malignancy-related mRNAs (c-myc and BCL-2). eIF4E activation may reflect increased eIF4E expression or phosphorylation of its inhibitory binding proteins (4E-BP). By immunohistochemical analyses of 148 tissues from 89 prostate cancer patients, we now show that both eIF4E expression and 4E-BP1 phosphorylation (p4E-BP1) are increased significantly, particularly in advanced prostate cancer versus benign prostatic hyperplasia tissues. Further, increased eIF4E and p4E-BP1 levels are significantly related to reduced patient survival, whereas uniform 4E-BP1 expression is significantly related to better patient survival. Both immunohistochemistry and Western blotting reveal that elevated eIF4E and p4E-BP1 are evident in the same prostate cancer tissues. In two distinct prostate cancer cell models, the progression to androgen independence also involves increased eIF4E activation. In these prostate cancer cells, reducing eIF4E expression with an eIF4E-specific antisense oligonucleotide currently in phase I clinical trials robustly induces apoptosis, regardless of cell cycle phase, and reduces expression of the eIF4E-regulated proteins BCL-2 and c-myc. Collectively, these data implicate eIF4E activation in prostate cancer and suggest that targeting eIF4E may be attractive for prostate cancer therapy.

Biochemical, pathologic and morphometric alterations induced in male B6C3F1 mouse liver by short-term exposure to dichloroacetic acid

Dichloroacetic acid (DCA) is a complete hepatocarcinogen and tumor promoter in the male B6C3F1 mouse. Published reports indicate that the compound is non-genotoxic. This study examines possible non-genotoxic (epigenetic) mechanisms by which DCA elicits its carcinogenic response. Correlative biochemical, pathologic and morphometric techniques are used to characterize and quantify the acute, short-term response of hepatocytes in the male B6C3F1 mouse to drinking water containing DCA. Cellularity, [3H]thymidine incorporation, DNA concentration, nuclear size, and binuclearity are evaluated in terms of level of exposure (0, 0.5 and 5 g/l) and length of exposure to DCA. The dose-related alterations in hepatocytes of animals exposed to DCA for 30 days or less indicate that short-term exposure to DCA results in inhibition of mitoses, alterations in cellular metabolism and a shift in ploidy class. Thus, DCA carcinogenesis may involve cellular adaptations, development of drug resistance and selection of phenotypically altered cells with a growth advantage.

Quantitative and qualitative immunohistochemical detection of myc and src oncogene proteins in normal, nodule, and neoplastic rat liver.

This study examined the possibility of using an immunohistochemical technique to detect the expression of myc and src oncogene proteins (ops) in livers of male Sprague-Dawley rats after treatment with the carcinogen diethylnitrosamine (with or without phenobarbital promotion) or untreated. We found that the majority of nodules and tumors from these livers stained for myc and src ops, indicating that myc and src expression did occur in these structures. These results were expected, since myc and src expression has been previously observed by others using different techniques. However, in our study, myc and src op staining was also noted in normal liver areas from rats in any of the four treatment groups (DENA, DENA + PB, PB alone, or untreated). The staining pattern of normal liver was different for each oncogene probe but was consistent within the four groups. In most cases, oncogene expression of normal liver occurred at sites of abnormal (but non-neoplastic) hepatocytes. The method reported here used both a qualitative technique of op expression analysis and a quantitative method using a Zeiss computer-driven image analysis system.

Quantitative image cytometry of hepatocytes expressing gamma-glutamyl transpeptidase and glutathione S-transferase in diethylnitrosamine-initiated rats treated with phenobarbital and/or phthalate esters.

Image cytometry was used to quantify the volume of liver expressing two histochemical markers associated with neoplasia, gamma-glutamyl transpeptidase (GGT) and the placental isozyme of glutathione S-transferase (GST-P). Rats were treated with diethylnitrosamine (DENA) followed by phenobarbital (PB), di(2-ethylhexyl)phthalate (DEHP), or di-n-octyl-phthalate (DOP) for 26 weeks. In one series, PB-treated rats were given 2.0%, 0.5%, or 0.1% DEHP in the feed. GGT expression was detected diffusely throughout the liver parenchyma in several treatment groups so that any enhanced expression in altered foci (AF) and nodules (N) was not apparent. GST-P was detected only in AF and N. GST-P may represent a second genetic alteration, as GST-P+ AF and N also expressed GGT but not the reverse. The peroxisome proliferator DEHP inhibited expression of GGT or GST-P in livers of either DENA-treated or DENA+PB-treated rats. With GST-P the reduction was correlated to a reduced number of AF and N. In contrast, DEHPs stereoisomer, DOP, was as effective as PB in promoting expression of both markers. We conclude that image cytometry of hepatocytes expressing GST-P can be used in the bioassay of the carcinogenic potential of chemicals that affect liver proliferation.

Abstract 4172: Overexpression of Cathepsin L is associated with prostate cancer development, progression, and reduced patient survival

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Although an estimated 217,730 men will develop prostate cancer in 2010, only 15% (32,050) will die of this disease. To avoid overtreatment, new biomarkers for early detection of potentially lethal prostate cancers are needed. Cathepsin L (Cat L), also known as the major excreted protein (MEP), is a cysteine proteinase capable of degrading the extracellular matrix. Increased levels of Cat L are found in ras transformed cells and in human prostate cancer cell lines (PC3, DU 145) and are associated with tumor cell invasion and metastasis in these experimental models. Since invasion and metastasis are the lethal aspects of cancer, we hypothesized that overexpression of Cat L will be associated with prostate cancer development and progression in men and with poor clinical outcome. Forty one prostate cancers (CaP), including all Gleason grades, and 62 specimens of benign prostate hyperplasia (BPH) were stained by immunohistochemistry in histologic sections of archival FFPE tissues for Cat L, Cat B and their endogenous inhibitors cystatin C and stefin A. Stain was evaluated according to intensisty (I) and % of epithelium stained (A). Semiquantitative observations were expressed as histoscores (HS = I × A). Histoscores were compared to proliferation (Ki67) and apoptosis (TUNEL) in adjacent histologic sections. Data were compared to five year recurrence and survival. The Institutional Review Board of St. Elizabeth Medical Center (Edgewood, KY) gave approval for this study. Expression of these cysteine proteinases and their inhibitors was confined to the epithelial portion of prostate tissues but was heterogeneous. Increased frequency of near uniform expression of Cat L was found in CaP relative to BPH from patients with or without CaP (p = 0.001) and was significantly correlated with cystatin C expression (p = 0.0044). The Cat L HS in CaP was 285% of that in BPH from patients without CaP, whereas the HS for Cat B was unchanged, suggesting that Cat L was the more predominant cysteine proteinase in neoplastic growth. Increased Cat L expression in neoplastic growth was not matched quantitatively by increased endogenous inhibitor expression. Cat L HS was significantly correlated with cell proliferation (p = 0.0001) and with apoptosis (p = 0.0180). Higher Gleason grade tumors had significantly higher Cat L HS as well as higher proliferation and apoptosis. Importantly, Cat L and proliferation, but not apoptosis, were significantly increased (p < 0.05) in CaP patients who recurred or died from CaP within 5 years of diagnosis compared to patients who survived without a recurrence. We conclude that Cat L is a significant factor in CaP development, progression and outcome and therefore is both a potential prognostic marker and target for CaP therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4172. doi:10.1158/1538-7445.AM2011-4172

Abstract 3451: Cell proliferation and apoptosis during chloroform-induced hepatocarcinogenesis in male F-344/N rats

The carcinogenic potential of chlorinated organics is of direct importance in human risk assessment. Most drinking water chlorinated organics are disinfection by products (DBPs) of water chlorination and many test positive in rodent bioassays. Trihalomethanes (THMs) are the most prevalent DBPs generated in chlorinated drinking water and chloroform (TCM) is the THM in highest concentration in finished drinking water. Human exposure to TCM occurs through ingestion of drinking water, inhalation and dermal exposure. TCM is carcinogenic to the liver and kidney of rodents including male and female B6C3F1 mice and male F-344/N and Osborne-Mendel rats. The carcinogenic mechanism of TCM in the rodent is not completely understood. Four mechanisms have been proposed: 1) mutagenicity; 2) reparative hyperplasia; 3) altered gene expression; and 4) secondary genotoxicity. Here, histopathology, immunohistochemistry, and quantitative image analysis were used to examine reparative cell proliferation and altered gene expression as potential mechanisms of TCM-induced hepatocellular carcinogenesis in male F-344/N rats. Animals were exposed to concentrations of 803 + 5 or 1592 + 21 mg/L in the drinking water for 78 or 100 weeks. Distilled water was the vehicle control. The high TCM dose increased the prevalence (% of animals with a lesion) of hepatocellular neoplasia (carcinoma and adenoma) 17.5% vs. 5.1% (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3451.