Robert P. C. Shiu

Pro-protein convertase gene expression in human breast cancer

As a first step towards elucidating the role that pro‐protein convertases play in the growth regulation of breast cancer, we studied the gene expression of 6 known human convertase members (PC1/PC3, PC2, furin/PACE, PACE4, PC5/PC6 and PC7/LPC) in human breast cancer tumors and cell lines. PC1, furin, PACE4 and PC7 mRNAs were detected by reverse transcriptase‐polymerase chain reaction (RT‐PCR) amplification in all 7 human breast cancer cell lines and 30 breast tumor tissues tested. PC5 expression was detected in 2/30 tumor tissues. PC2 mRNA, however, was not detected. In situ hybridization localized furin mRNA to the tumor cells; adjacent fibrous stroma and blood vessel elements were negative for furin gene expression. Thirty breast tumors with varying quantities of estrogen and progesterone receptors were assayed for furin, PACE4 and PC1 mRNAs by quantitative RT‐PCR, and 22 tumors were assayed for PC7 mRNA. An apparent association was observed only between PACE4 and estrogen receptors. No statistically significant correlation was found between the levels of steroid receptors and the expression of human furin, PC1 and PC7 genes. Convertase mRNA levels appeared similar in both the estrogen‐responsive and ‐unresponsive breast cancer cell lines. Also, pro‐protein convertase mRNAs were not detected in 9 histologically normal human breast tissues. These results suggest that elevated expression of some members of the pro‐protein convertase gene family is a characteristic of human breast cancer, an event which may be important for human breast tumorigenesis.Int. J. Cancer 71: 966‐971, 1997.

Estrogen Receptors α and β in Rat Decidua Cells: Cell-Specific Expression and Differential Regulation by Steroid Hormones and Prolactin1

Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogenbinding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), a and b. In this investigation, we analyzed the expression of decidual ERa and ERb, studied their regulation by PRL and steroid hormones and examined the ability of decidual ERb to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ERa and ERb by PRL and ovarian steroid hormones. Whereas PRL, estradiol, and progesterone all increased ERb messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ERa. Estradiol had no effect on ERa expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ERa and had no synergistic effect on ERb expression. The use of GG-AD cells, which express only ERb, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ERb is expressed in addition to ERa in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ERa, but concomitantly increases the expression of a functional ERb that mediates estradiol up-regulation of the decidual progesterone receptor. (Endocrinology 141: 3842‐3851, 2000)

Endocrine Communication between Conceptus and Mother: Placental Lactogen Stimulation of Maternal Behavior

The possible role of the conceptus in stimulating the onset of maternal behavior through its secretion of placental lactogens and their passage into the brain was investigated in female rats. In the first study, significant mitogenic activity in the Nb2 lymphoma cell bioassay was detected in cerebrospinal fluid (CSF) samples collected by push-pull perfusion from rats on days 12-21 of pregnancy, coincident with the establishment of placental function. In contrast, mitogenic activity was absent from CSF in lactating and gonadectomized, virgin females. In a second study the mitogenic activity in day 12 pregnant samples was neutralized 71% with antibodies to rat placental lactogen-I (rPL-I) and > 90% with a combination of antibodies to rPL-I plus rPL-II. In contrast, activity on day 21 of pregnancy, 1 day prepartum, was reduced by antibodies to rPL-II (> 85%), but not by antibodies to rPL-I, indicating that the predominant lactogen in the CSF prepartum is rPL-II. The behavioral actions of placental secretions were assessed in the third experiment by infusing recombinant rPL-I and purified rPL-II directly into the medial preoptic area of the brain of steroid-primed, nulliparous rats. Latencies to respond maternally to foster young were significantly reduced in rPL-I- and rPL-II-treated rats (2- to 3-day latencies) when compared with latencies in control females (5- to 6-day latencies). Thus, the conceptus through its secretion of rPLs which apparently gain access to the CSF helps to prime the pregnant females brain to respond maternally at the end of gestation. This endocrine communication between the developing conceptus and pregnant female appears to be an important part of the biological system which helps to establish successful maternal care.

The prolactin-inducible protein (PIP/GCDFP-15) gene: cloning, structure and regulation.

The androgen and prolactin responsive prolactin-inducible protein (PIP)/gross cystic disease fluid protein (GCDFP-15) is expressed in benign and malignant human breast tumors and in such normal exocrine organs as sweat, salivary and lacrimal glands. In this paper we report the cloning and structure of the human gene, and describe potential mechanisms involved in its regulation by hormones. The entire PIP gene, 7 kb long, was found in a single recombinant phage clone. The gene has 4 exons ranging from 106 bp to 223 bp in length. Nuclear run-on experiments utilizing PIP genomic fragments to detect nascent PIP transcripts revealed that both androgen and prolactin increased transcription of the PIP gene. Neither hormone had any effect on the stability of PIP precursor RNA or mature mRNA. Therefore the PIP gene is an excellent model by which to study the molecular events associated with the actions of prolactin and androgen in the regulation of gene expression in mammalian cells.

Basic fibroblast growth factor (BFGF) and two of its receptors, FGFR1 and FGFR2 : gene expression in the rat brain during postnatal development as determined by quantitative RT-PCR

Regional and temporal patterns of the expression of basic fibroblast growth factor (bFGF), and two of its high affinity receptors (FGFR1 and FGFR2), were examined in the male rat brain during early postnatal development; the reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain mRNA measurements which were expressed relative to mRNA for GAPDH as a constant. In the rat cerebrum, the mRNAs for bFGF and for FGFR2 were relatively low in amount within the first postnatal week, but by 28 days, they were as high as in the 1-year-old rat cerebrum. In contrast, the expression of FGFR1 was biphasic: mRNA levels were higher at postnatal days 1 and 28 than at day 21. Quantitation of mRNA from microdissected regions of 28-day-old rat brain revealed that the expression of bFGF and of FGFR2 showed a marked variation between regions but the expression of FGFR1 appeared less variable between the regions that were analyzed. For all three genes the hippocampus appeared to have high relative amounts of mRNA. The temporal patterns of expression of bFGF, FGFR1 and FGFR2 also differed with brain region during early postnatal development. In the occipital cortex and inferior colliculus, the mRNAs for bFGF and FGFR2 both increased in amount during the first month, unlike that for FGFR1. However, in the cerebellum, the highest expression of bFGF and FGFR1 mRNAs occurred at postnatal day 1; FGFR2 expression apparently showed less change with age. The temporal changes in bFGF, FGFR1 and FGFR2 expression in different brain regions during early postnatal development suggest that receptor regulation may permit different physiological effects of bFGF according to brain region and developmental age.

C-myc gene expression alone is sufficient to confer resistance to antiestrogen in human breast cancer cells

C‐myc is implicated in the initiation, progression and estrogen response of breast cancer. To further investigate the role of c‐myc in breast cancer, we have developed clonal MCF‐7 human breast cancer cell lines harboring a stably‐transfected human c‐myc gene, whose expression was stringently controlled by the bacterial reverse tetracycline transcription activator protein. The expression of the endogenous genomic c‐myc gene in MCF‐7 cells was abolished by the potent pure estrogen antagonist, ICI 182,780. Functional c‐Myc protein was identified by both Western immunoblotting and by its ability to transactivate a chimeric plasmid consisting of E‐box sequences upstream of the luciferase reporter gene. One MCF‐7 clone, 35im, was chosen for further characterization. C‐myc induction by doxycycline was rapid and dose dependent; c‐myc mRNA appeared as early as 30 min after doxycycline addition and stimulation of c‐myc expression required as little as 50 ng/ml doxycycline, with c‐myc mRNA levels reaching a plateau at 2.5 μg/ml doxycycline. ICI 182,780 or doxycycline (a tetracycline analog) treatment did not alter the mRNA levels of Max, the c‐myc binding partner. As in wildtype MCF‐7 cells, the growth of clone 35im was inhibited by 1 μM or less of ICI 182,780 and stimulated by 10 nM to 1 μM 17β‐estradiol. When maintained in a complete medium containing 5% normal fetal bovine serum (FBS) and ICI 182,780, doxycycline induced cell growth by 400% in an 8‐day assay. A similar level of growth was achieved with doxycycline treatment in cells that were arrested by the use of charcoal‐stripped FBS. Doxycycline had no effect on the growth of a control MCF‐7 clone (18c). Apoptosis, assessed by caspase‐dependent cleavage of poly(ADP‐ribose) polymerase, was unchanged in clone 35im cells after treatments with doxycycline or ICI 182,780. The present study demonstrates that c‐myc alone is sufficient to confer antiestrogen resistance in human breast cancer. Our novel c‐myc‐inducible MCF‐7 cell model offers a unique opportunity to study the diverse actions of the c‐myc proto‐oncogene in human breast cancer.

Potentiation of radiation sensitivity in breast tumor cells by the vitamin D3 analogue, EB 1089, through promotion of autophagy and interference with proliferative recovery

1,25-Dihydroxyvitamin D3 and vitamin D3 analogues, such as EB 1089, potentiate the response to ionizing radiation in breast tumor cells. The current studies address the basis for this interaction by evaluating DNA damage and repair, the effect of interference with reactive oxygen generation, the involvement of p53 and caspase-3, signaling through c-myc, as well as the induction of senescence and multiple modes of cell death. EB 1089 failed to increase the extent of radiation-induced DNA damage or to attenuate the rate of DNA repair. The reactive oxygen scavengers N-acetyl-l-cysteine and reduced glutathione failed to protect the cells from the promotion of cell death by EB 1089 and radiation. Whereas MCF-7 cells expressing caspase-3 showed significant apoptosis with radiation alone as well as with EB 1089 followed by radiation, EB 1089 maintained its ability to confer susceptibility to radiation-induced cell killing, in large part by interference with proliferative recovery. In contrast, in breast tumor cells lacking p53, where radiation promoted extensive apoptosis and the cells failed to recover after radiation treatment, EB 1089 failed to influence the effect of radiation. EB 1089 treatment interfered with radiation-induced suppression of c-myc; however, induction of c-myc did not prevent senescence by radiation alone or radiation-induced cell death promoted by EB 1089. EB 1089 did not increase the extent of micronucleation, indicative of mitotic catastrophe, induced by radiation alone. However, EB 1089 did promote extensive autophagic cell death in the irradiated cells. Taken together, these studies suggest that the effect of EB 1089 treatment on the radiation response is related in part to enhanced promotion of autophagic cell death and in part to interference with the proliferative recovery that occurs with radiation alone in p53 wild-type breast tumor cells. [Mol Cancer Ther 2006;5(11):2786–97]

Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to adriamycin.

Although the primary response to Adriamycin (doxorubicin) in p53 mutant MDA-MB231 and p53 null MCF-7/E6 breast tumor cells is apoptotic cell death, the residual surviving population appears to be in a state of senescence, based on cell morphology, beta galactosidase staining, induction of p21(waf1/cip1) and down regulation of cdc2/cdk1. Suppression of apoptosis in MDA-MB231 and MCF-7/E6 cells treated with Adriamycin using the broad spectrum caspase inhibitor, zvad-Fmk, results in substantial induction of autophagy. Overall sensitivity to Adriamycin, measured by clonogenic survival, is not altered in the cells undergoing autophagy, consistent with autophagy contributing to cell death in response to Adriamycin. The free radical scavengers, glutathione and N-acetyl cysteine attenuate the accelerated senescence response to Adriamycin in MCF-7 cells as well as in MDA-MB231 and MCF-7/E6 cells, but protect primarily the MCF-7 cells, indicating that reactive oxygen is unlikely to be directly responsible for Adriamycin toxicity in breast tumor cells. Expression of caspase 3 or induced expression of c-myc in MCF-7 cells fails to abrogate accelerated senescence induced by Adriamycin. Taken together, these studies suggest that accelerated senescence induced by Adriamycin is similar in cells with wild type p53 and in cells lacking functional p53 with regard to the upregulation of p21(waf1/cip1), down regulation of cdc2 and the involvement of reactive oxygen species. Furthermore, accelerated senescence, autophagy and apoptosis all appear to be effective in suppressing self-renewal capacity in breast tumor cells exposed to Adriamycin.

Expression of the rat BFGF antisense RNA transcript is tissue-specific and developmentally regulated.

The basic fibroblast growth factor (bFGF) gene locus is transcribed into a number of mRNA transcripts including an antisense mRNA derived from the opposite DNA strand of the bFGF gene. Expression of this natural antisense RNA has been implicated in regulation of the bFGF sense mRNA expression and turnover. In the present study we examined the developmental pattern of expression of the bFGF antisense transcript in fetal and postnatal rat tissues. Northern hybridization with a strand-specific cRNA probe detected a 1.5-kb polyadenylated antisense RNA in all tissues examined except brain, in which two transcripts were detected as a doublet of approximately 1.3-1.5 kb in size. The level of antisense transcript expression was markedly tissue- and age-dependent. In the developing brain, both sense and antisense transcripts were detected by Northern hybridization, but the pattern of their expression was inversely related. The 6.0-kb bFGF sense transcript increased in an age-dependent manner from days 3-30 of postnatal development while the antisense transcript decreased to nearly undetectable levels over the same period. In embryonic (days 15-19) liver, kidney, heart and intestine bFGF antisense RNA expression was low but increased dramatically at parturition, rising 5-10-fold over fetal levels by 10 days of age, then declined slowly to a new steady-state level in adult tissues. The level of antisense RNA in these tissues was much higher than that of bFGF sense mRNA, which was undetectable by Northern analysis. Sense and antisense trancripts were also detected in midgestational (11.5 days) embryos by RT-PCR. Antisense expression did not increase when embryos were explanted and cultured for 48 h (9.5-11.5 days). The apparent reciprocal relationship between the abundance of sense and antisense bFGF transcripts in developing brain supports the possibility of a regulatory role for the antisense transcript in this tissue. There was no evidence for a reciprocal relationship between sense and antisense expression in the other tissues examined, indicating that the relationship between sense and antisense RNA expression may be tissue-specific.

Progestin regulation of EGF-receptor mRNA accumulation in T-47D human breast cancer cells

Incubation of T-47D human breast cancer cells with the synthetic progestin, medroxyprogesterone acetate (MPA), resulted in a time and dose-dependent increase in epidermal growth factor-receptor (EGF-R) mRNA. Concentrations of MPA as low as 1 nM resulted in a greater than five fold increase in EGF-R mRNA. A significant increase (2-3 fold) in EGF-R mRNA was apparent 12 hr after exposure to MPA and a further increase was seen 12-48 hr after addition of MPA to the cultures. From these studies it is concluded that the increased EGF binding in progestin-treated T-47D cells results at least in part from increased EGF-R gene expression. We believe this is the first report of a steroid hormone modulating expression of this growth factor receptor gene.