Gloria M. Calaf

Critical role of oxidative stress in estrogen-induced carcinogenesis

Mechanisms of estrogen-induced tumorigenesis in the target organ are not well understood. It has been suggested that oxidative stress resulting from metabolic activation of carcinogenic estrogens plays a critical role in estrogen-induced carcinogenesis. We tested this hypothesis by using an estrogen-induced hamster renal tumor model, a well established animal model of hormonal carcinogenesis. Hamsters were implanted with 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethinylestradiol (αEE), menadione, a combination of αE2 and αEE, or a combination of αEE and menadione for 7 months. The group treated with βE2 developed target organ specific kidney tumors. The kidneys of hamsters treated with αE2, αEE, or menadione alone did not show any gross evidence of tumor. Kidneys of hamsters treated with a combination of αE2 and αEE showed early signs of proliferation in the interstitial cells. Kidneys of hamsters treated with a combination of menadione and αEE showed foci of tumor with congested tubules and atrophic glomeruli. βE2-treated tumor-bearing kidneys showed >2-fold increase in 8-iso-prostaglandin F2α (8-iso-PGF2α) levels compared with untreated controls. Kidneys of hamsters treated with a combination of menadione and αEE showed increased 8-iso-PGF2α levels compared with untreated controls, whereas no increase in 8-iso-PGF2α was detected in kidneys of αEE-treated group. A chemical known to produce oxidative stress or a potent estrogen with poor ability to produce oxidative stress, were nontumorigenic in hamsters, when given as single agents, but induced renal tumors, when given together. Thus, these data provide evidence that oxidant stress plays a crucial role in estrogen-induced carcinogenesis.

Characterization of ataxia telangiectasia fibroblasts with extended life-span through telomerase expression.

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar degeneration, immunodeficiencies, genomic instability and gonadal atrophy. A-T patients are hypersensitive to ionizing radiation and have an elevated cancer risk. Cells derived from A-T patients require higher levels of serum factors, exhibit cytoskeletal defects and undergo premature senescence in culture. We show here that expression of the catalytic subunit of telomerase (hTERT) in primary A-T patient fibroblasts can rescue the premature senescence phenotype. Ectopic expression of hTERT does not rescue the radiosensitivity or the telomere fusions in A-T fibroblasts. The hTERT+AT cells also retain the characteristic defects in cell-cycle checkpoints, and show increased chromosome damage before and after ionizing radiation. Although A-T patients have an increased susceptibility to cancer, the expression of hTERT in A-T fibroblasts does not stimulate malignant transformation. These immortalized A-T cells provide a more stable cell system to investigate the molecular mechanisms underlying the cellular phenotypes of Ataxia-telangiectasia.

Short-Chain Fatty Acids Inhibit Invasive Human Colon Cancer by Modulating uPA, TIMP-1, TIMP-2, Mutant p53, Bcl-2, Bax, p21 and PCNA Protein Expression in an In Vitro Cell Culture Model

High intakes of dietary fiber or resistant starches have been associated with a lower incidence of colon cancers. Because short-chain fatty acids (SCFA) such as butyrate are produced in the colonic lumen by the bacterial fermentation of dietary fibers and resistant starches, we hypothesized that SCFA may inhibit the development of invasive human colon cancers. To test this hypothesis, primary human invasive colonocytes were isolated from fresh surgical specimens and treated with 0.01 mol/L acetate, propionate or butyrate; cell invasion, cell adhesion, F-actin polymerization, urokinase plasminogen activator (uPA), tissue inhibitor matrix metalloproteinase (TIMP)-1, TIMP-2 and mutant p53, Bcl-2, Bax, p21 and proliferating cell nuclear antigen (PCNA) protein expression levels were examined. Although each of the SCFA tested significantly reduced primary cell invasion, butyrate was the most potent, inhibiting primary invasive human colon cancer invasion by 54% (P < 0.0001). The effects of SCFA on primary cell invasion appeared to be independent of cell adhesion and F-actin polymerization but dependent on the inhibition of uPA (P < 0.05) and the stimulation of TIMP-1 and TIMP-2 activities (P < 0.05). Protein expression levels of mutant p53, p21, Bax, Bcl-2 and PCNA were significantly altered by each of the SCFA tested (P < 0.05). These data indicate that SCFA inhibit invasive human colon cancer by modulating proteolytic uPA and antiproteolytic TIMP-1 and TIMP-2 activities, but their mechanisms of action on tumor suppression, apoptosis and growth arrest may differ.

TGFBI expression reduces in vitro and in vivo metastatic potential of lung and breast tumor cells

Controversy has arisen as to the role of transforming growth factor-β-induced protein (TGFBI) in the regulation of tumor metastasis. Using lung and breast cancer cell lines (H522 and MCF-7, respectively), we established that TGFBI induced cell adhesion to extracellular matrix proteins by activating adhesion-associated signaling and subsequent structure reformation, ultimately leading to cells less motile; whereas TGFBI reduced abilities of colony formation in soft agar, penetration through matrix gel, and activation of matrix metalloproteinases 2 and 9. Furthermore, injection of TGFBI-expressing cells into immuno-deficient mice resulted in a significant reduction in tumor metastasis in vivo. Taken together, these data suggest that TGFBI moderates the metastatic potential of cancer cells.

Profiling of Differentially Expressed Genes Induced by High Linear Energy Transfer Radiation in Breast Epithelial Cells

Methods to define patterns of gene expression have applications in a wide range of biological systems. Several molecular biological techniques are used to study expression patterns during the neoplastic progression of breast epithelial cells. In the present study, differential expression of human oncogenes/tumor suppressor genes in human breast epithelial cell lines irradiated with low doses of high linear energy transfer radiation and treated with estrogen was assessed with cDNA expression arrays. Transformed and tumorigenic cell lines were compared with the control cell line to identify differentially expressed genes during tumorigenic progression. Autoradiographic analysis showed that of the 190 genes analyzed, 49 genes showed a high level of altered expression, and 12 genes had minor differences in expression levels. Among these 49 genes, 17 genes were altered at all stages of transformation, 21 were altered only at the early stage, and the remaining 11 were at the late stage of transformation to the tumorigenic stage of progression. Among the 11 late stage–associated genes, seven genes were altered exclusively in the tumorigenic cell lines and in Tumor‐T. Of the 17 all‐stage genes, six were randomly selected, and we confirmed their altered expression by gene‐specific semiquantitative reverse transcription polymerase chain reaction, followed by Northern blot analysis. The results showed that the mRNA expression patterns of all these genes were consistent with the expression pattern seen on the array. Among these six genes, five genes, including c‐myc, puf, MNDA, c‐yes, and Fra‐1 showed upregulation, and the other gene, RBA/p48, showed downregulation in the transformed and tumorigenic cell lines compared with the control MCF‐10F cell line. Investigation of these genes should help establish the molecular mechanisms of progression that are altered by radiation and estrogen treatment. A number of candidates reported here should be useful as biomarkers involved in breast carcinogenesis.

Human RecQL4 Helicase Plays Critical Roles in Prostate Carcinogenesis

Prostate cancer is the second leading cause of cancer-associated deaths among men in the western countries. Here, we report that human RecQL4 helicase, which is implicated in the pathogenesis of a subset of cancer-prone Rothmund-Thomson syndrome, is highly elevated in metastatic prostate cancer cell lines. Increased RecQL4 expression was also detected in human prostate tumor tissues as a function of tumor grade with the highest expression level in metastatic tumor samples, suggesting that RecQL4 may be a potential prognostic factor for advanced stage of prostate cancer. Transient and stable suppression of RecQL4 by small interfering RNA and short hairpin RNA vectors drastically reduced the growth and survival of metastatic prostate cancer cells, indicating that RecQL4 is a prosurvival factor for prostate cancer cells. RecQL4 suppression led to increased poly(ADP-ribose) polymerase (PARP) synthesis and RecQL4-suppressed prostate cancer cells underwent an extensive apoptotic death in a PARP-1-dependent manner. Most notably, RecQL4 knockdown in metastatic prostate cancer cells drastically reduced their cell invasiveness in vitro and tumorigenicity in vivo, showing that RecQL4 is essential for prostate cancer promotion. Observation of a direct interaction of retinoblastoma (Rb) and E2F1 proteins with RecQL4 promoter suggests that Rb-E2F1 pathway may regulate RecQL4 expression. Collectively, our study shows that RecQL4 is an essential factor for prostate carcinogenesis.

Malignant transformation of human bronchial epithelial cells with the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Sufficient evidence has demonstrated that cigarette smoking is causally associated with various types of human cancers. In the United States, about 90% of deaths from lung cancer among men and 79% of those among women are associated with smoking. Tobacco‐specific nitrosamines are formed from nicotine and related tobacco alkaloids and are the most carcinogenic compounds of tobacco smoke. The most potent N‐nitrosamine contained in tobacco smoke is 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone, NNK. In the our study, the oncogenic transforming effects of graded doses of NNK were examined using papillomavirus‐immortalized human bronchial epithelial cells. Growth kinetics, saturation density, resistance to serum‐induced terminal differentiation, anchorage independent growth and tumorigenicity in nude mice were used to investigate the various stages of transformation in bronchial epithelial cells. We show here that immortalized human bronchial epithelial cells in culture can be malignantly transformed by treatment with NNK (100 μg/ml or 400 μg/ml) for 7 days. Transformed cells produced progressively growing subcutaneous tumors upon inoculation into nude mice. Immunofluorescence staining for keratin expression confirmed the epithelial nature of the tumor cells. Increased expression of p16, β‐catenin and PCNA in the established cell lines were detected by immunofluorescence staining and quantified by confocal microscopy. These data suggested that NNK can induce malignant transformation of human bronchial epithelial cells, and the tumor cell lines established are useful models in investigating the carcinogenic mechanism(s) of NNK.

Allelic imbalance at 11p15.5-15.4 correlated with c-Ha-ras mutation during radiation-induced neoplastic transformation of human breast epithelial cells

Breast cancer is the most frequent malignancy in women throughout much of the developed world and is associated with a multistage process involving a number of genetic mutations and their corresponding cellular phenotypic alterations. It has already been shown that neoplastic transformation of a spontaneously immortalized human breast epithelial (MCF‐10F) cell line by radiation, in combination with estrogen, represents a successful model in studying the molecular and biological alterations that may contribute to the tumorigenic process. In the present study, the incidence of allelic alterations (microsatellite instability/loss of heterozygosity) on chromosome 11 in different radiation‐induced primary and secondary tumorigenic cell lines, relative to the control MCF‐10F cells was investigated. We identified 3 regions of the chromosome 11 (11p15–p15.5, 11q13 and 11q23) that showed high incidence of LOH among these tumor cell lines and suggested a potential role for these chromosomal regions in breast carcinogenesis. Among them, locus 11p15.5, where c‐Ha‐ras oncogene is located, had incidence of allelic imbalance between 25–40%. Furthermore, direct sequencing analysis of codons 12 and 61 of the c‐Ha‐ras oncogene identified various point mutations. These data highlight the importance of chromosome 11 in radiation induced malignant transformation of human breast epithelial cells and suggest the usefulness of the model in uncovering specific derangements during breast cancer progression.

PAC1 is a direct transcription target of E2F-1 in apoptotic signaling.

E2F-1 controls multiple cellular activities through transcriptional regulation of its target genes. As a mediator of cell death, E2F-1 can eliminate latent neoplastic cells through apoptosis. However, the mechanism by which E2F-1 mediates cancer cell killing is largely unknown. In this paper, we report that phosphatase of activated cells 1 (PAC1) phosphatase is a direct transcription target of E2F-1 in signaling apoptosis. We show that ectopic E2F-1 increases expression of PAC1 at both transcriptional and translational levels in breast cancer cells. E2F-1 physically interacts with the promoter of PAC1, binds to its consensus sequence in the promoter and transactivates the PAC1 promoter. E2F-1 suppresses extracellular signal-regulated kinase (ERK) phosphorylation through PAC1 and causes cancer cell death by apoptosis following treatment with a chemotherapeutic agent N-4-hydroxyphenylretinamide (4-HPR). Furthermore, ectopic PAC1 inhibits ERK phosphorylation and mediates cell killing. Moreover, endogenous E2F-1 upregulates PAC1 and suppresses ERK activity, leading to cell death in response to 4-HPR. These results reveal a crucial role of PAC1 in E2F-1-directed apoptosis. Our study demonstrates that E2F-1 mediates apoptosis through transcriptional regulation of PAC1 and subsequent suppression of the ERK signaling. Our findings establish a functional link between E2F-1 and mitogen-activated protein kinases. The E2F-1–PAC1 cascade in cancer cell killing may provide a molecular basis for cancer therapeutic intervention.

Molecular alterations in tumorigenic human bronchial and breast epithelial cells induced by high let radiation

Carcinogenesis is a multi-stage process with sequence of genetic events governing the phenotypic expression of a series of transformation steps leading to the development of metastatic cancer. In the present study, immortalized human bronchial (BEP2D) and breast (MCF-10F) cells were irradiated with graded doses of either 150 keV/micrometer alpha particles or 1 GeV/nucleon 56Fe ions. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Cell fusion studies indicated that radiation-induced tumorigenic phenotype in BEP2D cells could be completely suppressed by fusion with non-tumorigenic BEP2D cells. The differential expressions of known genes between tumorigenic bronchial and breast cells induced by alpha particles and their respective control cultures were compared using cDNA expression array. Among the 11 genes identified to be differentially expressed in BEP2D cells, three (DCC, DNA-PK and p21(CIP1)) were shown to be consistently down-regulated by 2 to 4 fold in all the 5 tumor cell lines examined. In contrast, their expressions in the fusion cell lines were comparable to control BEP2D cells. Similarly, expression levels of a series of genes were found to be altered in a step-wise manner among tumorigenic MCF-10F cells. The results are highly suggestive that functional alterations of these genes may be causally related to the carcinogenic process.