extfieldsofstudy: Biology Medicine

text:
Autocrine regulation of cell proliferation by estrogen receptor-alpha in estrogen receptor-alpha-positive breast cancer cell lines

Background Estrogen receptor-α (ERα) is essential for mammary gland development and is a major oncogene in breast cancer. Since ERα is not colocalized with the cell proliferation marker Ki-67 in the normal mammary glands and the majority of primary breast tumors, it is generally believed that paracrine regulation is involved in ERα mediated cell proliferation. In the paracrine model, ERα-positive cells don't proliferate but will release some paracrine growth factors to stimulate the neighboring cells to proliferate. In a subpopulation of cancer cells in some primary breast tumors, however, ERα does colocalize with the cell proliferation marker Ki-67, suggesting an autocrine regulation by ERα in some primary breast tumors. Methods Colocalization of ERα with Ki-67 in ERα-positive breast cancer cell lines (MCF-7, T47D, and ZR75-1) was evaluated by immunofluorescent staining. Cell cycle phase dependent expression of ERα was determined by co-immunofluorescent staining of ERα and the major cyclins (D, E, A, B), and by flow cytometry analysis of ERαhigh cells. To further confirm the autocrine action of ERα, MCF-7 cells were growth arrested by ICI182780 treatment, followed by treatment with EGFR inhibitor, before estrogen stimulation and analyses for colocalization of Ki-67 and ERα and cell cycle progression. Results Colocalization of ERα with Ki-67 was present in all three ERα-positive breast cancer cell lines. Unlike that in the normal mammary glands and the majority of primary breast tumors, ERα is highly expressed throughout the cell cycle in MCF-7 cells. Without E2 stimulation, MCF-7 cells released from ICI182780 treatment remain at G1 phase. E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERα and Ki-67 as well as the cell cycle progressing through the S and G2/M phases. Inhibition of EGFR signaling does not inhibit the autocrine action of ERα. Conclusion Our data indicate that ERα can mediate estrogen-induced cell proliferation in an autocrine mode in ERα-positive breast cancer cell lines. All of the three ERα-positive cell lines used in our study showed colocalization of ERα and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

Background
The ovarian-derived steroid hormone estrogen is one of the main regulators of mammary epithelial cell proliferation and differentiation. While estrogen is required for normal mammary gland development, cumulative exposure to estrogen during a woman's lifetime is a high risk factor for breast malignancy. The action of estrogen is exerted by binding and activating the estrogen receptors, ERα and ERβ. Studies on the expression patterns of estrogen receptors and gene knock-out mouse models indicate that ERα is the primary estrogen receptor for mammary epithelial cell proliferation and differentiation. While ERα is expressed exclusively in the epithelial cells, ERβ is expressed in both epithelial and stromal cells but the expression of ERβ in the epithelial cells is very weak [1][2][3][4].
In ERβ knock-out mice, the mammary glands can still form the bronchoalveolar structure and lactate normally [5,6]. In ERα knock-out mice, however, the mammary gland remains at rudimentary duct stage without further development [7][8][9]. While the role of ERβ in breast cancer is less understood, ERα is one of the most known oncogenes in breast cancer [10][11][12]. Abnormal expression of ERα is found in about 70-80% of human breast cancers, and about 50% of ERα-positive breast cancer patients respond to anti-estrogen therapy [10,13]. When overexpressed in the mammary gland epithelial cells of transgenic mice, ERα leads to mammary malignancy [14,15].
Despite the significant role of ERα in mediating estrogeninduced cell proliferation in normal mammary development and breast cancer, the mechanism is still not fully understood. In the normal mammary gland, ERα expression is found only in a subpopulation of the mammary epithelial cells and the percentage of ERα-positive cells is affected by the physiologic conditions. Unlike most other cellular receptors, ERα expression is not found in proliferating mammary epithelial cells [1][2][3][16][17][18]. This observation leads to the prevailing concept that ERα mediates cell proliferation in a paracrine manner [1,19]. In this paracrine model, ERα-positive cells don't proliferate; the ERαpositive cells, however, when stimulated by estrogen, will produce and release paracrine growth factors, which in turn stimulate the neighboring cells to proliferate [1,9]. The EGF family member amphiregulin could be one of those important paracrine growth factors involved in ERα-mediated mammary gland epithelial cell proliferation [20]. It is not clear why ERα-positive cells cannot proliferate, or why ERα does not stimulate cell proliferation in an autocrine mode or in both autocrine and paracrine modes. A recent study by Cheng et al. challenged the paradigm that ERα cannot mediate ERα-positive cell proliferation [17]. The authors proposed that ERα is degraded early in the cell cycle, which accounts for the dissociation of the ERα-staining and cell proliferation marker.
In contrast to the mechanism in normal mammary tissues, ERα might mediate cancer cell proliferation via paracrine and/or autocrine modes in primary breast cancers. In ERα-positive primary breast cancers, the percentage of ERα-staining cells varies from 10% to 50% or higher [1,3,21,22]. Most of the ERα-staining cells are not stained with Ki-67 in these ERα-positive primary breast tumors [1,3,23,24]. In some primary breast tumors, however, ERα does colocalize with the cell proliferation marker Ki-67 in a sub-population of tumor cells; the percentage of ERα and Ki-67 duel-staining cells varies among different patients, from 0% to ~5% of total cells [1,3]. The co-staining of ERα and Ki-67 suggests that ERα-positive cells in these primary breast tumors might be capable of proliferating, i.e., ERα might be able to mediate cell proliferation via autocrine fashion in these primary breast tumor cells.
Using breast cancer cell line models, ERα has been shown to have non-genomic effect in addition to the traditional genomic action [25]. In its genomic action as a transcriptional factor, ERα activation in MCF-7 cells can induce the expression of cyclin D and myc to promote cell cycle progression [26][27][28][29]. When stimulated by estrogen, the effect on cell proliferation in MCF-7 cells is usually not obvious until 5-7 days after the stimulation [30][31][32]. It is not clear why the impact is delayed for such long time and that raises the question whether the activation of ERα alone is sufficient to drive cell cycle progression through all the phases for mitosis. For the non-genomic action of ERα, cell signaling initiated from the cell surface can activate multiple pathways such as the ERK and AKT pathways [33][34][35]. Using the EGFR inhibitor, Levine and colleagues demonstrated that EGFR is required for the cell surface ERα-activated signaling transduction [35]. Marks and colleagues demonstrated that inhibition of the MAPK and PI3K-AKT pathways can prevent estrogen-induced mitogenesis in MCF-7 cells [36]. Considering all of the information derived from different perspectives, it is very likely that MCF-7 cells and some other ERα-positive breast cancer cell lines might be regulated by ERα via the autocrine as well as the paracrine modes. In this study, we demonstrated that ERα is colocalized with Ki-67 in MCF-7, T47D, and ZR75-1 cells, the ERα-positive breast cancer cell lines used in our study. Using MCF-7 cell line, we demonstrated that ERα is present in all the phases of cell cycle and activation of ERα in G1 phase promotes cell cycle progression through mitogenesis, supporting the autocrine mode of regulation. Finally, we demonstrated that EGFR activation is not required for the autocrine regulation of cell proliferation by ERα.  19) were obtained from Santa Cruz (Santa Cruz, CA). Antibodies for ERK1/2 (137F5), phospho-ERK1/2 (E10), AKT, phospho-AKT/Ser473, and EGFR were obtained from Cell Signaling Technology (Danvers, MA). Secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes, Inc, Eugene, OR) was used for green fluorescent staining, secondary antibody conjugated with Rhodamine Red-X (Jackson ImmunoResearch, West Grove, PA) was used for red fluorescent staining. Heat inactivated FBS and Charcoal-Dextrin stripped FBS (CD-FBS) were obtained from Omega (Tarzana, CA). Estrogen was obtained from Sigma (St. Louis, MO). ICI182780 was obtained from TOCRIS (Ellisville, MO). Gefitinib was obtained from LC Laboratories (Woburn, MA). EGF was obtained from Invitrogen (Carlsbad, CA).

Cell culture and treatments
DMEM/F12 medium (MediaTech Inc, Herndon, VA) containing 10% heat inactivated FBS and 5 μg/ml insulin was used for routine maintenance of the three ERα-positive breast cancer cell lines, MCF-7, T47D, and ZR75-1 (ATCC). For experimental assays, phenol-red free DMEM/ F12 (MediaTech Inc, Herndon, VA) containing 5% CD-FBS was used unless otherwise specified. To arrest cells in G1 phase by ICI182780, cells were seeded in culture dishes for 1-2 days to let the cells reach about 30-40% confluency before replacement with medium containing ICI182780. To evaluate the effect of estrogen stimulation, cells were stimulated by adding E2 either after removal of ICI182780 or in the presence of ICI182780. For estrogen stimulation after removal of ICI182780, cells treated with ICI182780 were washed briefly two times with serum-free phenol red-free DMEM/F12 to remove ICI182780, followed by replacement with medium containing 5 nM estrogen but without ICI182780. The control cells were replaced with medium containing vehicle only. For estrogen stimulation in the presence of ICI182780, cells were treated with 10 nM ICI182780 for various times before adding ten fold of E2 (100 nM) to the cells without removing ICI182780. ICI182780 stock solution was made at 10 mM in DMSO, then serially diluted in DMSO to 10 μM before diluted in culture medium. Estrogen stock solution was made at 10 mM in ethanol, then serially diluted in ethanol to 10 μM before diluted in culture medium. To inhibit the EGFR activation, cells were treated with Gefitinib for 2 hr first; without removing Gefitinib, estrogen or EGF was then added to the cells for stimulation. Gefitinib stock was made in DMSO at 1-50 mM, the stock was diluted in culture medium to make the final concentrations at 1-50 μM.

Immunofluorescent staining
Cells grown on glass coverslips were briefly washed with ice-cold Dulbecco's PBS and fixed with cold 4% parafor-maldehyde containing 0.01% Triton X-100 for 25 min on ice. After washing once briefly with PBS-T (Dulbelcco's PBS containing 0.01% Triton X-100), the cells were treated with 0.05% Triton x-100 in PBS for 10 min for permeabilization. The cells were washed once briefly with PBS-T, followed by blocking in PBS-T containing 5% normal serum and 50 mM NH4Cl for 30 min at room temperature. The cells were then incubated with primary antibodies diluted in PBS-T containing 1% normal serum. Anti-ERα antibody was diluted at 1:50, anti-Ki-67 at 1:100, anti-cyclin D at 1:150, anti-cyclin E at 1:100, anticyclin A at 1:100, and anti-cyclin B at 1:100. For the antibodies with blocking peptides available, the antibodies (ERα, cyclin A, cyclin E) were evaluated for its binding specificity by incubation with 5 fold of blocking peptides before applying to cells for primary antibody incubation. After overnight incubation at 4°C, the cells were washed four times with PBS-T, 7 min each time. Then the cells were incubated for 1 hr at room temperature with fluorescent dye-conjugated secondary antibodies (1:200) in PBS-T containing 1% normal serum. The cells were washed 4 times with PBS-T, 7 min each time before mounted on slides with VectoShield with DAPI (Vector Laboratories, Burlingame, CA). Immunofluorescent staining(s) was observed under Olympus BX50 Fluorescence Microscope with Optronics MagnaFire digital camera (Microscope Image Center, UVM).

Flow cytometry analysis
For analysis of cell cycle distribution, cells were harvested by trypsinization, pelleted by centrifugation, and washed once with Dulbecco's PBS. While vortexing cells, ice-cold 70-75% ethanol was added drop wise to resuspend the cells. The cells were fixed in 70% ethanol for at least 1 hr at 4°C before staining or stored at -20°C freezer. Before staining, 1-3 millions of cells were pelleted, washed once with PBS, followed by resuspension in 1 ml PBS containing 20 μg/ml PI (propidium iodide), 150 μg/ml RNaseA, 10 mM EDTA, and 0.1% Triton X-100. Incubation was carried in the dark at 37°C for 15-30 min. For the analysis of cell cycle distribution of ERα-staining cells, cells were harvested by trypsinization and the cell number counted. The cells were pelleted by centrifugation (2000 rpm, 5 min, 4°C). After resuspension with culture medium, 3 million of cells were transferred into Ependorff tube and pelleted by centrifugation (500 × g, 4°C, 2 min). The cell pellet was resuspended in 1 ml PBS-T containing 1% paraformaldehyde and cells were fixed on ice for 10 min. The cells were pelleted by brief centrifugation, resuspended in PBS containing 0.05% Triton-X100 for permeabilization at room temperature for 5 min. After brief washing, cells were incubated with 5% normal serum in PBS-T (30 min, room temperature) for blocking. Cells were pelleted and resuspended in PBS-T containing anti-ERα (F10, 1:100 dilution) and 0.5% BSA, followed by incubation at 4°C for overnight. The cells were washed 4 times with PBS-T, 7 min each, by centrifugation and resuspension, followed by incubation with the secondary antibody anti-mouse Alexa Fluor 488 (1:300) at room temperature for 40 min. After washing with PBS-T for 4 times, 7 min each, the cells were incubated with 0.5 ml PI-RNaseA solution for 30 min at 37°C. Flow cytometry analysis was performed using the Coulter Epics XL-MCL (VCC core facility, UVM). About 20,000 cells were analyzed for each sample.

Colocalization of ER and Ki67 in ER -positive breast cancer cell lines
ERα is not colocalized with the cell proliferation marker Ki-67 in normal mammary cells, and colocalization of ERα and Ki-67 is rare even in primary breast tumors [1][2][3][16][17][18]23]. To study the mechanism of how cell proliferation is mediated by ERα, the ERα-positive breast cancer cell lines were used in our study. In all the three ERα-pos-itive cell lines evaluated in this study, i.e., MCF-7, T47D, and ZR75-1, a large proportion of cells showed duel-staining of ERα and Ki-67 (Fig. 1). The colocalization of ERα with Ki-67 was evaluated in cells under different culture conditions and the duel-staining was observed in all culture conditions, including cell culture in regular DMEM/ F12 supplemented with 10% FBS, phenol-red free DMEM/F12 supplemented with charcoal dextrin stripped FBS, with and without insulin, with and without estrogen. While these data don't exclude the possibility of a paracrine regulation of cell proliferation by ERα in these cells, the colocalization of ERα and Ki-67 does support the hypothesis that ERα may mediate cell proliferation in an autocrine mode in these ERα-positive breast cancer cell lines, a mechanism absent in normal mammary cells and most primary breast tumors [1][2][3][16][17][18]23,24].

Deregulated expression of ER in all phases of cell cycle
In normal breast tissue, ERα is expressed in quiescent (G 0 ) cells or early G 1 phase [1,17]. In MCF-7 cells, activation of ERα leads to cyclin D1 and myc expression, indicating the expression of ERα in G1 phase [26]. Ki-67 is a proliferation marker expressed in late G1, S, G2, and M phases of the cell cycle [39]. In these ERα-positive breast cancer cell lines, most Ki-67-staining cells contained high levels of ERα suggesting that ERα might be present throughout the cell cycle progression in these cell lines (Fig. 1). To address this possibility, we evaluated the colocalization of ERα with the cyclins for different phases of cell cycle (cyclin D for G1 phase; cyclin E for late G1 phase; cyclin A for G1/ S/G2 phases; cyclin B for M phase) [40][41][42] ing cells (Fig. 2A). Furthermore, flow cytometry analysis demonstrated that MCF-7 cells with high ERα expression (ERα high ) were distributed across G0/G1, S, and G2/M phases (Fig. 2B). These data indicate that ERα expression during cell cycle progression is deranged in MCF-7 cells.

E2 stimulation of ICI182780 treated cells induces expression of ER and Ki-67
While the distribution of ERα across all phases of cell cycling supports the autocrine mode of action in cell proliferation, it also raises the possibility that the colocalization of ERα with Ki-67 might be a coincidence. To further determine whether ERα can mediate cell proliferation in an autocrine mode, we sought to arrest the cells with the pure ERα antagonist ICI182780 followed by estrogen stimulation. When treated with ICI182780 for 72-96 hrs, no cell was observed with high ERα expression and only a few cells were stained with Ki-67, indicating that both ERα and Ki-67 were down-regulated by ICI182780 (Fig. 3). Consistently, ICI182780 treatment leads to cell cycle arrest at G1 phase (Fig. 3). The inhibitory effect of ICI182780 was observed similarly at concentrations from 10 nM to 50 nM, therefore 10 nM was selected for our later experiments.
To evaluate the effect of estrogen stimulation, cells were stimulated by adding E2 either after removal of ICI182780 or in the presence of ICI182780. We first evaluated the effect of estrogen stimulation after removal of ICI182780. MCF-7 cells were treated for ICI182780 for 72-96 hr and the treatment was stopped by washing with fresh serum-free medium to remove ICI182780. The cells were then stimulated with estrogen by adding medium containing 5 nM estrogen but without ICI182780. The control group of cells was replaced with medium without estrogen. In the cells stimulated with estrogen, both ERα and Ki-67 were induced with ERα and Ki-67 colocalized in most cells (Fig. 4A). In the control group of cells without estrogen stimulation, no high level of ERα or Ki-67 was observed although the cells were released from ICI182780 treatment. We also determined the effect of estrogen stimulation by adding estrogen directly to the cells still under ICI182780 treatment (Fig. 4B). The continuous presence of ICI182780 was to help minimize the secondary factors that might promote cell proliferation. Cells were treated with 10 nM ICI182780 for 72-96 hrs before the estrogen was added to the cells for a final concentration of 100 nM. Similar to the result shown in Fig.  4A, excessive estrogen stimulation in the presence of ICI182780 also induced the expression of high levels of ERα and Ki-67 with the two proteins colocalized in most cells (Fig. 4B). Collectively, these data indicate that activation of ERα by estrogen in ICI182780 arrested cells induces the expression of high levels of ERα and Ki-67 and their colocalization, supporting the autocrine mode of ERα action.

E2 stimulation releases cell cycle arrest by ICI182780 to promote cell cycle progression
We next examined the effect of estrogen stimulation on cell cycle progression. As described above, cells were stimulated with estrogen either after release from ICI182780, or estrogen was added at 10 fold higher concentration (100 nM E2) directly to cells without removing ICI182780 (10 nM ICI). Similar results were observed for either stimulation (Fig. 5, and data not shown). In cells stimulated with estrogen after release from ICI182780, most cells were still in the G1 phase until 24 hr after stimulation. At 44 hr after estrogen stimulation, about 45% of the cells were in S or G2/M phases, indicating that activation of ERα promotes the progressing of the cell cycle (Fig.  5). Interestingly, induction of high levels of ERα and Ki-67 appeared much earlier, with high levels of ERα appeared at around 6 hr. It is noteworthy that high levels of ERα appeared earlier than Ki-67, supporting that the activation of ERα is mediating the expression of Ki-67 and the cell cycle progression. These data indicate that the colocalization of ERα and Ki-67 is not a coincidence and support the hypothesis that activation of ERα can promote cell proliferation by autocrine regulation.

EGFR signaling is not required for the autocrine regulation of cell proliferation by ER
In the paracrine regulation of cell proliferation by ERα, EGFR signaling is required [20]. Therefore we evaluated whether EGFR signaling is required for the autocrine regulation of cell proliferation by ERα in MCF-7 cells. EGFR inhibitor Gefitinib was used to block EGFR signaling, 20 μM of Gefitinib was chosen based on our preliminary experiments using a wide range concentrations of Gefitinib (1 μM -50 μM) (Fig. 6D, and data not shown). We first evaluated whether Gefitinib treatment inhibits E2 stimulation induced expression and colocalization of ERα and Ki-67. MCF-7 cells were treated first with 10 nM ICI182780 for 72-96 hr, followed by adding the EGFR inhibitor Gefitinib to a final concentration of 20 μM for 2 hr, then by adding estrogen to a final concentration of 100 nM. In the presence of ICI182780 and Gefitinib, estrogen stimulation still induced the expression and colocalization of ERα and Ki-67 (Fig. 6B). Conceivably, Gefitinib treatment does not inhibit estrogen stimulation induced cell cycle progression (Fig. 6C lower panel). In the control groups of MCF-7 cells treated with ICI182780 for 72-96 hr, ERα and Ki-67 were down-regulated and the cells remained at G1 phase (Fig. 6A, C upper panel). In the growth curve studies and the EGF stimulation experiments, Gefitinib at 20 μM is sufficient to block EGFR signaling but not toxic to the cells (Fig. 6D, and data not shown). From these data, we concluded that EGFR signal-Deregulated expression of ERα in the cell cycle in MCF-7 cells Figure 2 Deregulated expression of ERα in the cell cycle in MCF-7 cells. A, co-immunofluorescent staining for ERα (green fluorescence) and the major cyclins (D, E, A, and B) (red fluorescence). Cells were immunofluorescent stained with antibody for ERα and the antibody for one of the cyclins, followed by secondary antibody. Colocalization was shown by overlaying (Merge). Magnification, 200×. B, Flow cytometry analysis for the cell cycle distribution of ERα high cells (cells with high levels of ERα). Cells were stained with antibody for ERα followed by Alexa Fluor 488, and nuclear DNA was stained with propidium iodide (PI). ing is not required for the autocrine action of ERα in mediating cell proliferation in MCF-7 cells.

Discussion
ERα is the primary estrogen receptor mediating the effects of estrogen in normal mammary epithelial cells [1][2][3][5][6][7][8][9]43]. In breast cancer, abnormal expression of ERα is found in about 70% of primary tumors [10,13]. Despite the significant role of ERα, the mechanism of how ERα mediates cell proliferation is not fully understood. The dissociation of ERα with cell proliferation markers such as Ki-67 in the normal mammary gland leads to the paracrine model of ERα-mediated cell proliferation. While colocalization of ERα and Ki-67 is rare in most primary breast cancers, there do exist some cancer cells in some primary breast tumors showing colocalization of ERα and Ki-67 suggesting the possibility of autocrine regulation. In this paper using the ERα-positive breast cancer lines, we presented data showing that most Ki-67 staining cells expressed high levels of ERα and that cells with high levels of ERα are distributed across different phases of cell cycle. We further demonstrated that activation of ERα in ICI182780 arrested cells can induce the expression and colocalization of high levels of ERα and Ki-67, and more importantly, the progressing of cell cycles from G1 phase to the G2/M phases. Collectively, our data support the hypothesis that ERα can mediate cell proliferation in an autocrine mode in these ERα-positive breast cancer cell lines.
Whether ERα mediates cell proliferation by autocrine and/or paracrine regulation is related to whether ERα-positive cells can proliferate or not. Inferred from the paracrine model is that ERα-positive cells are a separate subpopulation of cells that don't proliferate, or even further that ERα-positive cells cannot proliferate because ERα inhibits the proliferation of ERα-positive cells [1,3,44,45]. Although ERα is known to promote cell proliferation in ERα-positive breast cancer cell lines such as MCF-7, exogenous overexpression of ERα in some cell lines such as MDA-MB-231 and MCF-10A does inhibit cell proliferation [46][47][48][49][50]. The mechanism of how ERα mediates cell proliferation may have clinical implication for breast cancer etiology and therapy. Malignant transformation is a multistep process that involves different combination of genes at different stage of malignancy [51]. Distinct sets of ERα-target genes might be involved for the autocrine versus paracrine regulation and it will be interesting to examine whether the resistance to endocrine-related therapy is correlated with the mode of ERα action.
All the three ERα-positive cell lines used in our study showed colocalization of ERα and Ki-67. Since the number of established ERα-positive breast cancer cell lines is much less than expected from the expression frequency of ERα in primary breast tumors, we speculated that ERα-positive cell lines could only be established from cancer cells with autocrine regulation in the primary tumors [13,52]. In that case, what is known about the breast cancer from the studies using the ERα-positive breast cancer cell lines may not apply to all the ERα-positive primary breast tumors. It needs to point out that although our data support the autocrine mode of ERα action, it remains to determine whether both autocrine and paracrine modes of action by ERα are involved in these ERα-positive breast cancer cell lines. ERα is expressed in both the nucleus and cell surface in MCF-7 cells [53,54]. Based on the levels of membrane ERα, MCF-7 cells can be separated into two sub-populations, mERαhigh and mERα low [55,56]. The levels of nuclear ERα also showed heterogeneity in MCF-7 cells. It is not clear whether the heterogeneity of nuclear ERα levels is caused by the differential expression of ERα during cell cycle progression or by the heterogeneous sub-populations of cells. As demonstrated in ICI182780 treated cells, even extremely low or basal level of ERα can mediate some of the cellular response to estrogen. To determine whether paracrine regulation is involved or not, a subline of MCF-7 with the ERα being completely shut off, i.e., eliminating the low basal level of ERα, will be needed to test their proliferation in response to estrogen when mixed with normal MCF-7 cells.
Together with the systemic hormones, growth factors are involved in mammary cell proliferation. EGFR signaling is essential for ERα-mediated cell proliferation in the normal mammary glands [20]. In MCF-7 cells, EGFR is involved in the non-genomic action of ERα and blocking of some EGFR activated pathways could inhibit ERαmediated cell proliferation [33][34][35][36]. Our data, however, indicate that EGFR signaling is not required for the autocrine regulation of cell proliferation by ERα.

Conclusion
ERα can mediate estrogen-induced cell proliferation in an autocrine mode in ERα-positive breast cancer cell lines. All of the three ERα-positive cell lines used in our study Time course of E2 induced expression of ERα and Ki-67 and cell cycle progression EGFR signaling is not required for the autocrine regulation of cell proliferation by ERα Figure 6 EGFR signaling is not required for the autocrine regulation of cell proliferation by ERα. A, MCF-7 cells treated with 10 nM ICI182780 for 72-96 hr, showing the down-regulation of ERα and Ki-67 by ICI182780 treatment. In A and B, ERα and Ki-67 were evaluated by immunofluorescent staining, nuclei were stained with DAPI. ERα-staining and Ki-67 staining were overlayed to show colocalization (Merge). Magnification, 400×. B, EGFR inhibitor Gefitinib does not inhibit E2 induced ERα and Ki-67 expression. MCF-7 cells were stimulated with 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72-96 hr, followed by adding the EGFR inhibitor Gefitinib to a final concentration of 20 μM for 2 hr, before adding estrogen to a final concentration of 100 nM for 16 hr. C, flow cytometry analysis of cell cycle progression, showing E2 stimulated cell cycle progression is not inhibited by Gefitinib. Upper panel, MCF-7 cells treated with ICI182780 for 72 hr. The cells were arrested at G1 phase. Lower panel, cell cycle progression was stimulated by 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72 hr, followed by treatment with the Gefitinib (20 μM) for 2 hr, before stimulation with 100 nM E2 for 36 hr. D, immunoblotting of phosphorylated ERK1/2, showing the inhibition of EGFR signaling by 20 μM Gefitinib. MCF-7 cells were serum starved for 24 hr, followed by treatment with 20 μM Gefitinib for 2 hr, before EGF stimulation for 5 min. The control group of cells was not treated with Gefitinib. Phosphorylation of ERK1/2 was used to assess the activation of the EGFR signaling.
showed colocalization of ERα and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation. Considering that the number of established ERα-positive breast cancer cell lines is much less than expected from the percentage of ERα-positive primary breast tumors, we speculated that ERα-positive cell lines could only be established from cancer cells with autocrine regulation in the primary tumors. In that case, what is learnt about the breast cancer from the studies using the ERα-positive breast cancer cell lines may not apply to all the ERα-positive primary breast tumors.


extfieldsofstudy: Psychology Medicine

text:
Does prevention for Alzheimer’s disease exist?

The prevention of Alzheimer’s disease is a growing public health concern amidst an ageing population. Meanwhile, there is no effective or curative treatment available where prevention could greatly reduce health costs. This review was based on reports of potential preventive factors, including modifiable lifestyle factors, as well as preventive pharmacological strategies. Although the present review was not systematic, the reports selected from PubMed using “Alzheimer’s disease” and “prevention” as key-words, allow us to affirm that pursuing a healthy lifestyle; physical, cognitive, leisure activities; good social engagement; a high consumption of fish, low consumption of dietary fat and moderate consumption of wine, and control of vascular risk factors appear to be potential factors for delaying dementia.

Delaying the onset of dementia syndrome is a serious health issue which has financial, economic, and social repercussions. The global prevalence of dementia was estimated at 24.3 million in 2001,¹ while that of Alzheimer's disease (AD) was 26.55 million in 2006, and is predicted to rise to 106.2 millions by 2050. Retarding the clinical picture of dementia by 12 months represents a huge reduction of around 9.2 million cases.
In Brazil and Latin America, the prevalence of dementia in subjects aged 65 years or older is 7.2% and 15.7% among non-illiterates and illiterates, respectively.² Some risk factors for developing AD are well established: age, family history, female gender (not for all surveys), low educational level, depressive symptoms, traumatic brain injury, the presence of E4 allele of APOE, hypercholesterolemia, smoking, sedentary life-style, and mild cognitive impairment.
Studies have established some factors associated to maintenance of good cognitive aging. In a longitudinal follow-up of a cohort of elderly females over a 15-year period, Barnes et al. observed that persons who remained cognitively stable presented a lesser number of clinical co morbidities (such as arterial hypertension and diabetes), less complaints regarding difficulties in activities of daily living, and greater social integration.³ More recently, in an eight year follow-up study of 3075 elders aged between 70 and 79 years in 1997, 30% of the sample had no cognitive decline, while multivariate analysis found the following baseline variables to be associated with being a maintainer: lower age, white race, higher educational and literacy levels, weekly moderate to vigorous exercise and non-smoker status. 4 Autopsy series of elderly subjects with normal cognitive function have found high burdens of pathological lesions associated with AD, for which several explanations have been proposed. One explanation points to compensatory mechanisms of brain reserve. In the study by Erten-Lyons et al., some factors associated with resistance to dementia despite high AD pathology were determined by comparing individuals with high burden pathology and normal cognition against patients with AD presenting an equivalent burden of lesions. In multiple regression analysis, hippocampal and total brain volumes were significantly larger in the group with normal cognitive functions. The authors proposed three possible explanations: larger brain volumes may indicate a greater preexisting brain reserve; the cognitively intact group may have had other forms of compensation or protection from the pathologic processes; and, the brain volume loss in the AD group may not have been directly caused by neurofibrillary tangles and senile plaques. The underlying protective process remains unknown. 5 The aim of this report was to describe some results of interventions intended to prevent conversion from healthy or mild cognitive impairment subjects into AD. Some interventions can modify outcome in terms of developing dementia for a certain period of time. Epidemiologic research has revealed various possible preventive factors, including modifiable lifestyle factors and pharmacological strategies. Our aim was to present the results of several clinical trials demonstrating efficacy in delaying progression to dementia. Only results from recent longitudinal studies, randomized controlled trials, and reviews assessing the prevention of AD, conversion of MCI to dementia will be described, where only PubMed articles with full-text in the English language published from 1998 to 2009 (June) using Alzheimer's Disease and prevention as key-words were selected.

Non-pharmacological interventions
Cognitive, leisure, and physical activities Some studies have found a positive association between leisure activities and lower risk for developing AD or dementia in general. 6,7 Verghese et al. have attempted to control for possible confounding factors such as the preclinical stage of dementia (which prevents elderly from performing leisure and cognitive activities). They evaluated community-dwelling subjects aged 75 to 85 years old during a period spanning from 1980 to 2001. Subjects were analyzed regarding participation in cognitive activities (reading books or newspapers, writing for pleasure, doing crossword puzzles, playing board games or cards, participating in group discussions, and playing musical instruments) and for physical activities (playing tennis or golf, swimming, bicycling, dancing, participating in group exercises, playing team games, walking for exercise, climbing more than two flights of stairs, doing housework, or babysitting). At the end of the follow-up (median time of 5.1 years), 124 subjects had developed dementia (AD, n=61; vascular dementia, n=30). Subjects who became demented tended to be older, had less schooling, and performed less cognitive activities (which reduce risk of AD, vascular dementia and mixed dementia), but not physical activities (with the exception of dancing). Among cognitive activities, reading, playing board games, and playing musical instruments were associated with a lower risk of dementia. 8 In a new analysis of this same sample performed to verify the influence of leisure or physical activity participation on risk of developing amnestic mild cognitive impairment (aMCI), the authors observed that cognitive activities were associated with lower risk of aMCI. 9 In a study of twin pairs of male veterans (Duke Twin Study of Memory in Aging), cognitive activity reduced development of dementia in 26% (particularly among homozygotic pairs -30% reduction). Physical activity however, played no modifying role. 10 Social activity is associated with risk for AD, with subjects that have low social engagement being more prone to developing dementia, while those exhibiting mid and late-life social activity were associated with greater cognitive and physical health. [11][12][13][14] Cognitive activities are associated with greater and preserved cognitive reserve, that is, the capacity of the brain to cope with the pathology of dementia through structural or functional characteristics and networks, or alternative cognitive strategies that protect the subject until a certain threshold is reached.
More recently, in a 24-week randomized controlled clinical trial, physical activity was associated with a lower risk for developing MCI and dementia among adults with subjective memory impairment. The study was conducted at the Royal Perth Hospital (Australia) in volunteers aged 50 years or older, randomly assigned to an education program or to a 24-week home-based program of physical activity. There was a significant, albeit modest difference in cognitive performance among those undertaking the physical program, a difference which persisted over an 18month follow-up period. 15 The literature on associations among physical activity, cognition, and risk for dementia is conflicting, but this latter study appears to demonstrate a clear positive effect.
In animal studies, physical activity stimulates angiogenesis, synaptogenesis, and neurogenesis. Rats performing treadmill running activity had more astrocytes and neuroblasts with proliferative ability in the subgranular zone of the dentate gyrus of the hippocampus, as well as an increased number of neurons in the transient stage that control rats. 16 Physical exercise also decreases risk factors for vascular diseases, and can release hormonal factors that boost neuronal functions, reinforcing the theoretical basis of potential benefits.

Alcoholic beverages
There is a large body of evidence suggesting that moderate alcohol consumption is associated with reduced risk of coronary heart disease, ischemic stroke and dementia. [17][18][19][20] Nineteen longitudinal cohort studies compared relative risk factors for either cognitive decline or dementia in older moderate alcohol consumers versus abstainers/never drinkers. In these studies, approximately 54% of the risk factors were significantly lower in moderate consumers. In a Swedish study, women were followed for 34 years, and analysis was made verifying different types of beverages (wine, beer, and spirits). Wine was protective for dementia, and the association was strongest among women who consumed wine only, and among smokers. Notwithstanding, consumption of spirits at baseline was associated with slightly increased risk of dementia. 21 Protective mechanisms involved in the association of alcohol consumption with decrease in risk of dementia and vascular alterations, include increase of HDL and decrease of LDL, resulting in lowering of risk due to cholesterol; reduction of insulin resistance and increase in insulin sensitivity; reduction of blood homocysteine; decrease of systemic arterial pressure; reduction in platelet aggregation and fibrinogen levels, with increased fibrinolysis; and reduction in inflammatory markers. Reduction of dementia is associated not only with lower vascular factors, but also with antioxidant polyphenols such as resveratrol. There appears to be an antagonist effect on NMDA receptors by alcohol, and anti-amyloidogenic activity by resveratrol. It has no effect on βe γsecretases, but instead promotes intracellular degradation of Aβ via a mechanism that involves the proteasome 20,22,23 . Although mild to moderate consumption (one to two drinks, <30 g/d) is associated with lower risk for cerebrovascular and cardiac diseases; intake of three or more drinks increases rate of ischemic and hemorrhagic stroke.

Mediterranean diet
The Mediterranean diet is characterized by high intake of fish, vegetables, legumes, fruit, cereal, and unsaturated fatty acids (olive oil); low intake of dairy products, meat, and saturated fatty acids, and moderate consumption of alcohol. Some studies have verified a protective factor of high Mediterranean diet adherence against MCI development (borderline reduction risk) and a reduced risk of MCI conversion to AD. [24][25][26]

Ginkgo Biloba
Recently, a randomized double blind placebo controlled trial (G biloba 240 mg/d versus placebo) was published involving five centers in the USA, conducted between 2000 and 2008, with a median follow-up of 6.1 years. Subjects were evaluated every 6 months and 523 individuals developed dementia (246 from placebo group versus 277 from G biloba group). Results have shown that Ginkgo Biloba provided no benefit in reducing AD development or conversion of MCI into AD 27 .

Statins
Four large cohort studies showed the same effect of reduced risk of incident AD with statin use. [28][29][30][31] Some doubts existed over which type of statin was most linked to decreased incidence of dementia based on their lipophilicity. Lipophilic statins were thought to pass the blood-brain barrier (BBB) more efficiently than hydrophilic statins. Haag et al. evaluated the use of cholesterol-lowering drugs and divided them into three classes: lipophilic statins (simvastatin, atorvastatin, cerivastatin), hydrophilic statins (pravastatin, fluvastatin, rosuvastatin), and non-statin cholesterol lowering drugs (fibrates, bile acid binding resins or nicotinic acid and derivatives). Use of statins, despite their lipophilicity, but not use of non-statin cholesterol lowering drugs, was associated with a reduced risk of AD compared with never users. 31 This protection was effective independent of APOE4 genotype status. These results seem to indicate that permeability of the BBB is not the main protection mechanism. Other mechanisms must be associated to reduced risk of dementia by statins: inhibition of cholesterol synthesis, endothelial functioning, atherosclerosis and oxidative stress reactions, inhibition of amyloid synthesis and reduction of neurofibrillary tangle burden. 31

Antihypertensive drugs
There is relatively strong evidence that hypertension in midlife represents a risk factor for dementia or cognitive decline. Recently, a neuropathologic study reported that persons who were treated for hypertension in midlife were less demented clinically and had less AD pathology than both hypertensive patients who were not treated and nonhypertensive patients. 32 The association of dementia with late-life blood pressure has yielded heterogeneous results. The effects of antihypertensive treatment in late life on risks of dementia are not uniform. It is known however, that arterial hypotension in late life can result in increased risk for cognitive impairment, through ischemia and hypoperfusion. A recent study has shown antihypertensive use to be associated with a significant reduction in dementia in subjects less than 75 years old. 33 Some randomized controlled trials of antihypertensive medications have shown reductions in incident dementia. [34][35][36] Doubts over response to specific classes of antihypertensive drugs remain.

Cholinesterase inhibitors
The use of cholinesterase inhibitors (donepezil, rivastigmine, galantamine) is well established for AD treatment, with relative increase or preservation of cognitive scores and improvements in measures of global clinical impression observed in studies involving patients with mild to moderate and moderate to severe stages of dementia. Mild cognitive impairment (MCI) is a recognized risk factor for AD development, particularly in amnestic presentation. Some studies, majority of which produced negative results, have been conducted to determine reduction in conversion of MCI to AD.

Donepezil
During a mean follow-up of three years, results showed no decrease in risk for conversion of MCI to AD upon use of donepezil compared to placebo, although a reduced risk for APOE4 carriers was identified. 37 In a later sub-analysis of the same sample, a lessened benefit in conversion to dementia was observed among individuals with concomitant depression. 38 In a randomized double blind placebo controlled clinical trial, with a follow-up of 48 weeks, a discrete improvement in scores on the modified ADAS-Cog (primary measure of efficacy) was found among MCI patients versus the placebo group. Nevertheless, no differences were observed in measures of global or functional impairments. A higher number of subjects in the active group abandoned treatment due to adverse events; 38 a similar result was demonstrated by Salloway et al. 39

Rivastigmine
The InDDEx study was a double blind, randomized, placebo-controlled trial of up 48 months which sought to investigate whether rivastigmine delayed progression to AD in patients with MCI, and to assess possible benefits among cognitive functions in MCI patients. Subjects were assigned to a rivastigmine group (n=508) or placebo group (n=510). No significant difference in rates were found in either dementia or cognitive benefit, along with more adverse events in the active group. 40

Galantamine
In two studies involving 2048 subjects who were allocated to placebo or active groups, galantamine failed to prevent the progression of MCI to dementia. These studies showed no significant changes in ADAS-Cog/MCI or ADCS/MCI scores at month 12 or month 24, irrespective of number of ApoE-4 alleles. Adverse events were more common in the galantamine group. 41 Current evidence is insufficient to recommend the prescription of cholinesterase inhibitors for MCI or as a measure to delay development of dementia. Several points however should be emphasized: a significant percentage of subjects with MCI, spontaneously revert to normal cognition, and not all cases of MCI progress to dementia; amnestic MCI subjects are more prone to convert to AD than non-amnestic MCI; the instruments available probably lack the sensitivity to measure an effect of these types of drugs.

Conclusions
Although our review was not systematic, several conclusions can be drawn. Having a healthy lifestyle; physical, cognitive, leisure activities; good social engagement; high consumption of fish, low consumption of dietary fat and moderate consumption of wine; control of vascular risk factors, such as arterial hypertension, diabetes mellitus, dyslipidemias, and being a non-smoker seem to be potential advantages in delaying dementia. These factors are all modifiable in the lives of individuals, and can lead to improved quality of life for all. Other bio and genetic markers may prove useful for determining who could most benefit from these interventions, as well as other pharmacological interventions.


text:
Relative contributions of chromatin and kinetochores to mitotic spindle assembly

It's the kinetochores, not the DNA, that initiate spindle assembly.

Introduction
Several microtubule-organizing mechanisms cooperate during mitotic spindle assembly in vertebrates (O'Connell and Khodjakov, 2007;Kaláb and Heald, 2008). These include direct "search and capture" of kinetochores by dynamic microtubules emanating from centrosomes (Kirschner and Mitchison, 1986) as well as nucleation and stabilization of microtubules in the vicinity of chromosomes (Carazo-Salas et al., 1999;Kaláb et al., 1999). Complete removal of centrosomes via laser irradiation or conventional micromanipulation does not prevent the formation of a functional bipolar spindle Hinchcliffe et al., 2001). Furthermore, recent computer models demonstrate that unbiased search and capture is not efficient enough to assemble a bipolar spindle in the typical time for human cells of 10-15 min (Wollman et al., 2005). Thus, microtubule nucleation and organization near the chromosomes appears to be a principal driver of spindle assembly even in centrosomal cells. This mechanism is thought to rely on the activity of RanGTP, which induces liberation of microtubule nucleation factors sequestered in the complex with importins. The Ran guanine nucleotide exchange factor (RanGEF) RCC1 converts RanGDP to RanGTP in the vicinity of chromatin, whereas Ran GTPase-activating protein (RanGAP) promotes GTP hydrolysis in the cytoplasm. Together, these activities establish a gradient of RanGTP centered on the chromatin (Kaláb et al., 2002, and it is generally assumed that microtubule nucleation and stabilization are promoted evenly in the volume of cytoplasm immediately surrounding the chromosomes (Bastiaens et al., 2006;Clarke and Zhang, 2008). Indeed, long strings of chromatin-coated beads in Xenopus laevis extracts exhibit microtubule nucleation and polymerization along the entire surface (Gaetz et al., 2006). In contrast, microtubules have been observed growing primarily at or near the primary constriction and rarely along the chromosome arms in cultured cells (Khodjakov et al., 2003;Maiato et al., 2004;Tulu et al., 2006). Thus, the centromere/ kinetochore acts as a "hot spot" of microtubule nucleation within the gradient of RanGTP. This pathway is stimulated D uring mitosis and meiosis in animal cells, chromo somes actively participate in spindle assembly by generating a gradient of Ran guanosine tri phosphate (RanGTP). A high concentration of RanGTP promotes microtubule nucleation and stabilization in the vicinity of chromatin. However, the relative contributions of chromosome arms and centromeres/kinetochores in this process are not known. In this study, we address this issue using cells undergoing mitosis with unreplicated genomes (MUG). During MUG, chromatin is rapidly separated from the forming spindle, and both centro somal and noncentrosomal spindle assembly pathways are active. MUG chromatin is coated with RCC1 and establishes a RanGTP gradient. However, a robust spindle forms around kinetochores/centromeres outside of the gradient peak. When stable kinetochore microtubule attachment is prevented by Nuf2 depletion in both MUG and normal mitosis, chromatin attracts astral micro tubules but cannot induce spindle assembly. These results support a model in which kinetochores play the domi nant role in the chromosomemediated pathway of mi totic spindle assembly.
To better understand the relative contributions of the centrosome, chromatin, and centromeres/kinetochores toward spindle assembly, we turned to human cells undergoing mitosis with unreplicated genomes (MUG) as a model (Brinkley et al., 1988). In these cells, kinetochores with a minimal amount of centromeric DNA become spatially separated from the bulk of chromatin, allowing us to discern the relationships between, kinetochores, chromatin, and the RanGTP gradient. by the chromosomal passenger complex (CPC) at the centromere, which is probably involved in Aurora B-mediated microtubule stabilization (Sampath et al., 2004). RanGTP is required for kinetochore-mediated microtubule organization (Tulu et al., 2006;Torosantucci et al., 2008), although it is not clear whether its distribution in a gradient affects the ability of the centromere/kinetochore to serve as an efficient microtubule organizer. A recent study demonstrated that spindles can assemble without a RanGTP gradient in Xenopus egg extracts. However, assembly proceeds only when the CPC, which is In agreement with previous studies in live (Khodjakov et al., 2003;Tulu et al., 2006) and fixed cells (Witt et al., 1980), kinetochores were consistently found at or near the foci of noncentrosomal microtubules after nocodazole washout. It should be noted that, shortly after the removal of nocodazole, spatial separation between kinetochores and the chromatin was not pronounced. Thus, we cannot rule out the possibility that factors emitted by the chromatin are required for microtubule nucleation in the vicinity of the kinetochores. However, microtubules were rarely seen forming in association with the chromatin that was not intertwined with kinetochores ( Fig. 1 C, inset), suggesting that kinetochores are needed for efficient microtubule nucleation during MUG.
Thus far, our data demonstrate that microtubules can be nucleated at noncentrosomal sites during MUG. However, MUG cells lack the integration of kinetochores and chromatin into a normal, unified mitotic chromosome. This raises the question of whether a functional spindle can assemble purely via centrosome-independent pathways in this system. It has been shown that laser ablation of centrosomes does not prevent formation of a functional bipolar spindle in several types of vertebrate somatic cells La Terra et al., 2005). We confirmed this observation by ablating both centrosomes in untreated centrin1-GFP-expressing HeLa cells during

Results and discussion
The centrosome-independent spindle assembly pathway is active during MUG A unique feature of MUG is that the majority of unreplicated kinetochores achieve alignment at the metaphase plate, whereas the bulk of chromatin becomes spatially separated from the kinetochores and pushed to the periphery of the cell ( Fig. 1 A). Exclusion of the chromatin from the spindle occurred rapidly upon nuclear envelope breakdown (NEB; Fig. 1 B). A few microtubules were consistently seen extending from the spindle poles toward the peripheral chromatin in fully assembled MUG spindles ( Fig. 1 A, arrows). However, centrosomes and the main body of the spindle, which contains the majority of the microtubules, were always centered at kinetochores. This suggests that kinetochores play a larger role in the process of spindle assembly compared with chromatin.
To establish whether or not cells retain a centrosomeindependent spindle assembly pathway during MUG, hydroxyurea (HU)-arrested centrin1-GFP-expressing HeLa cells were treated with 5 µM nocodazole for 30 min followed by washout to allow microtubule regrowth. Cells were subsequently fixed and stained for DNA, kinetochores, and microtubules. Significant numbers of noncentrosomal microtubules were observed in addition to those emanating from the centrosomes (Fig. 1 C). all fixed cells revealed that there were significant numbers of microtubules, accounting for the clear regions observed in DIC ( Fig. 2 and Fig. S1). In two of the fixed cells, the spindle was clearly bipolar with well-focused poles. Similar to control spindles, polar regions in acentrosomal spindles during MUG contained the pole-organizing protein NuMA (nuclear-mitotic apparatus protein; Fig. 2 F). In another cell, NuMA localization was poorly defined, and a bipolar organization of microtubules was lacking (Fig. S1 F). We conclude that (a) the normal chromosome/centromere/kinetochore architecture is not an absolute prerequisite for the centrosome-independent spindle assembly and (b) kinetochores are the predominant contributors to this process. Our observations that centrosome-independent mechanisms for spindle formation are strong during MUG prompted us to examine the distribution of RanGTP relative to the spindle, chromosomes, and kinetochores. First, we compared the distribution of the RanGEF RCC1 between normal HeLa mitosis and MUG. In control cells, RCC1 coated the entire mass of chromosomes, and its distribution during metaphase resembled a disk positioned G2 (Fig. 2 A). In all cases (n = 5), cells entered mitosis within 6 h after ablation and formed transient monopolar spindles, which subsequently bipolarized ( Fig. 2 B, 0:05 and 1:15). The chromosomes achieved proper congression, and cells then exited mitosis after a moderate delay (3.0 ± 2.7 h).
To test whether centrosome-independent spindle formation occurs during MUG, we ablated both centrosomes in HU-arrested HeLa cells (Fig. 2 C). The GFP intensity of daughter centrioles increases before MUG entry and is similar to the accumulation of centrin at the daughter centriole in normally cycling cells as they progress from S through G2. This feature was used as a prognostic factor to identify cells that were about to enter MUG. Cells that entered MUG in the absence of centrosomes also assembled spindles. Six cells, which were followed through the entire duration of MUG by time-lapse differential interference contrast (DIC) microscopy, exited slightly faster than controls under the same conditions (2.5 ± 1.5 h; Fig. 2 D). Timely exit from MUG indicates that all kinetochores become attached to microtubules and the mitotic checkpoint is satisfied. We have previously shown that this checkpoint is active during MUG (O'Connell et al., 2008). To determine the organization of microtubules during MUG without centrosomes, several additional cells were fixed and processed for immunofluorescence microscopy 30 min after entry into MUG. Tubulin staining in A view rotated by 60° around the y axis shows a uniform coating through the entire volume at which the mitotic spindle assembles. (B) During MUG, RCC1 is also bound to chromatin but is pushed to the side by the spindle that assembles around kinetochores. This is most evident as a hole created by spindle microtubules passing through the chromatin visible in the rotated view. (C) Although antibodies against RCC1 recognize small pieces of equatorial chromatin, centromeres/kinetochores labeled with CENP-A-GFP lack RCC1 staining (insets). Bars, 5 µm.
undergoing MUG showed a dramatic drop in RanGTP levels perpendicular to the spindle axis at the equator (Fig. 4, compare B with D, red line). Even though some small chromatin fragments congressed at the metaphase plate, no detectable hot spots of RanGTP were visible there. Unfortunately, in our conditions, Rango-2 exhibited rapid photobleaching, a property precluding 3D reconstruction of the RanGTP gradient during MUG. Extrapolating from the distribution of chromatin and RCC1, the gradient likely had a toroidal shape as well. Together with timelapse imaging showing the rapid exclusion of chromatin after NEB ( Fig. 1 B), these data demonstrate that MUG spindles preferentially assemble at kinetochores outside the area of maximum RanGTP concentration.
RanGAP catalyzes the hydrolysis of GTP bound to Ran and is critical for maintenance of the directionality of nuclear transport. During mitosis, RanGAP is recruited to kinetochores (Joseph et al., 2002). Although there is no direct evidence that kinetochore-associated RanGAP1 is active, it does raise the possibility that the concentration of RanGTP within the microenvironment around kinetochores is lower as the result of locally increased RanGAP1 activity. RanGAP1 is also present on kinetochores of HeLa cells during MUG, although the staining is of low intensity, as it is in control cells (Fig. S3,  A and B). Quantification of fluorescence intensities demonstrates that there is not a statistically significantly difference (P > 0.05) in RanGAP1 levels on kinetochores between mitosis and MUG (Fig. S3). The preferential incorporation of kinetochores into spindles and the marginalization of chromatin during MUG demonstrate that kinetochores remain active microtubule organizers even when they reside outside the peak of the RanGTP gradient. The morphology of spindles during MUG makes it an attractive system for future studies relating to RanGTP microenvironments. at the equator (Fig. 3 A). During MUG, RCC1 was predominantly bound to peripheral chromatin, often forming a toroid around the axis of the spindle (Fig. 3 B). Although there were some small pieces of chromatin within the central spindle that also possessed RCC1, kinetochores were consistently devoid of this protein (Fig. 3 C).
RCC1 is the principal source of RanGTP. Therefore, the concentration of RanGTP should be highest in the vicinity of the chromatin that becomes distributed to the periphery of the spindle during MUG. To directly test this prediction, we visualized the gradient of RanGTP via a fluorescence resonance energy transfer (FRET) biosensor, Rango-2 (Kaláb and Soderholm, 2009). Rango-2 was transiently transfected into wild-type HeLa cells before treatment with HU. Increased FRET between YPet and CyPet signals cargo liberation induced by the binding of RanGTP to importins and correlates with high local concentration of free RanGTP produced by RCC1.
During normal mitosis in HeLa cells, the maximum concentration of RanGTP in the cell corresponds to the position of both the chromatin and centromeres/kinetochores. As a result, FRET was high and relatively uniform when plotted across the spindle equator (Fig. 4, A and B; and Fig. S2 A). When measured along the spindle axis, RanGTP levels gradually decreased toward the spindle poles, forming a gradient, as previously reported .
Rango-2 also displayed a higher level of FRET around chromatin during MUG (Fig. 4, C and D; and Fig. S2 B), confirming that RanGTP is generated and induces release of cargo from importins in this system. As predicted by RCC1 immunostaining, the concentration of RanGTP in MUG was maximal near peripheral chromatin, which was identifiable in DIC by its rough granular appearance (Fig. S2 C). Consistent with MUG spindle morphology and in contrast to normal mitosis, cells kinetochores inevitably reside in close spatial proximity to the source of RanGTP (i.e., chromatin). As a result, the distribution of astral microtubules is obscured by the kinetochore fibers (K fibers). The exclusion of chromatin from the spindle during MUG provides a unique advantage for visualizing potential biases in astral microtubule growth (Fig. S3 C). This advantage can be extended by elimination of K fibers. To this end, we depleted Nuf2, a component of the microtubule-binding Ndc80 complex which is required for stable attachment of microtubules to the kinetochore (DeLuca et al., 2002). siRNA depletion reduced Nuf2 levels to 20-25%, as determined by quantifying the amount of Hec1, a protein in complex with Nuf2, on kinetochores.
We find that in the absence of K fibers, most astral microtubules grow toward the chromatin during MUG. In cells with

The RanGTP gradient guides microtubule growth toward chromatin
In Xenopus extracts, the RanGTP gradient serves a dual role. It promotes microtubule nucleation (Carazo-Salas et al., 1999;Kaláb et al., 1999) and guides microtubule growth toward the chromatin (Dogterom et al., 1996;Carazo-Salas and Karsenti, 2003;Athale et al., 2008). Less is known about the specific functions of the gradient in somatic cells, although it is clear that the gradient facilitates mitotic spindle assembly . It has been suggested that guiding the growth of centrosomegenerated microtubules toward the chromosomes would significantly accelerate search and capture (Wollman et al., 2005). Formally, the RanGTP gradient can provide such guidance. However, the effects of RanGTP on the growth of astral microtubules in cells have not been directly investigated because kinetochores outside the peak of the RanGTP gradient could not be assessed. We demonstrate that chromatin in MUG is fully competent to liberate microtubule nucleation and stabilization factors through a gradient of RanGTP. However, the role of chromatin appears to be limited to a biasing of search and capture of centrosomal microtubules. Microtubule organization at the kinetochores plays a primary role in chromosome-mediated spindle assembly even when kinetochores are not supported by the maximum RanGTP concentration.

Materials and methods
Cell culture and microscopy Wild-type and centrin1-GFP (Piel et al., 2000)-, CENP-A (centromere protein A)-GFP (O'Connell et al., 2008)-, and H2B-monomeric RFP (mRFP)/-tubulin-GFP (Steigemann et al., 2009)-expressing HeLa cells were maintained in DME at 37°C in an atmosphere of 5% CO 2 . For live cell imaging, coverslips were mounted in Rose chambers containing CO 2independent medium (Invitrogen) and kept at 37°C with a heated enclosure. MUG was observed 40 h after mitotic shake off and treatment with 2 mM HU (Sigma-Aldrich; O'Connell et al., 2008). For depolymerization of microtubules, 5 µM nocodazole was added to the culture medium for a minimum of 30 min. Microtubule regrowth was initiated with two 7.5-ml changes of drug-free DME. HU-treated cells were incubated with 100 nM Hesperadin (a gift from T. Kapoor, The Rockefeller University, New York, NY) for >12 h before fixation.
5D time-lapse imaging of -tubulin-GFP and H2B-mRFP was performed on a spinning disk confocal system (Revolution; Andor Technology) coupled to a microscope (TE2000-E; Nikon). Z series (0.5-µm steps) were obtained with a 100× NA 1.49 Apochromat total internal reflection fluorescence oil immersion objective lens, and maximum intensity projections were made with ImageJ software (National Institutes of Health). For fixed cells, z series (0.2-µm steps) were collected on a DeltaVision system (Applied Precision) with a 100× NA 1.35 UPlan-Apochromat oil immersion objective (Olympus) and subsequently deconvolved using SoftWoRx 2.5 software (Applied Precision). Quantification of RanGAP signal was performed by calculating the integrated pixel intensities in 9 × 9-and 13 × 13-pixel squares centered at a kinetochore. Background and final kinetochore intensity were then calculated as previously described (O'Connell et al., 2008).

RNAi
HeLa cells were shaken off and plated on coverslips and allowed to attach for 1 h before transfection of a siRNA oligonucleotide specific for human Nuf2 (DeLuca et al., 2002) using Oligofectamine (Invitrogen) according to the manufacturer's instructions. At this point, 2 mM HU was also added. An equally effective strategy to deplete Nuf2 in MUG was to transfect the oligonucleotide 6 h before shake off and HU addition. As a control, transfections were performed with Oligofectamine alone.

Immunofluorescence
To costain kinetochores, tubulin, and DNA, cells were washed once with warm PEM (100 mM Pipes, pH 6.9, 2.5 mM EGTA, and 5 mM MgCl 2 ) and extracted with 0.5% Triton X-100 in prewarmed PEM for 1-2 min. This was followed by fixation for 10 min in 4% paraformaldehyde diluted in PEM. The same procedure was used to stain for kinetochores and Ran-GAP1. For immunofluorescence of cells after laser ablation, the fixation and extraction steps were combined. For RCC1 staining, cells were fixed and permeabilized for 20 min on ice in a 1:1 mixture of methanol and acetone chilled to 20°C. For Hec1 and tubulin staining, cells were extracted in warm PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl 2, pH 7.0) for 2 min followed by fixation in PHEM with 4% paraformaldehyde and 0.1% glutaraldehyde for 10 min. Primary antibodies and dilutions used in this study were 1:200 monoclonal anti--tubulin (clone DM 1A; Sigma-Aldrich), 1:4,000 rat monoclonal anti--tubulin (clone YL1/2; Novus Biologicals), 1:10,000 human CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) antiserum (a gift from B. Brinkley, Baylor College of Medicine, Houston, TX), 1:50 polyclonal anti-RCC1 (Millipore), 1:350 monoclonal anti-RanGAP1 (Invitrogen), and 1:1,000 monoclonal anti-Hec1 (a gift from C. Walczak, Indiana University, Bloomington, IN). a significant amount of chromatin intervening between the centrosomes, this can give the appearance of a spindle (Fig. 5 B). In some cells, the chromatin clustering toward one side is even more pronounced. In these cases, the space between separated centrosomes, where a spindle would be, is almost completely devoid of microtubules (Fig. 5 C), and kinetochores/centromeres are scattered throughout the area occupied by microtubules (Fig. S3 E). Such a microtubule and chromatin distribution in Nuf2-depleted cells is not unique to MUG. Normal mitotic cells depleted of Nuf2 with chromosomes biased toward one side also lack a spindle intervening between centrosomes (Fig. 5 D). It is important to note that this morphology was never observed in control depletions and is unlikely to represent an early stage of mitosis or MUG. Therefore, the RanGTP gradient guides microtubules toward the chromatin during mitosis in somatic cells.
Our three experimental conditions, MUG, acentrosomal MUG, and Nuf2-depleted MUG, allowed us to test various combinations of centrosomes, kinetochores, and chromatin for their ability to form a mitotic spindle. Although chromatin and kinetochores usually work together toward their integration into the spindle, when separated, the kinetochores are a dominant factor, as indicated by the formation of a functional spindle in cells with a mispositioned chromatin/RanGTP gradient during MUG. It is possible that the kinetochore-mediated mechanism is enhanced by the microtubule-nucleating activity of the CPC (Sampath et al., 2004;Maresca et al., 2009), which is enriched at the equator with kinetochores during MUG (O'Connell et al., 2008). However, we found that inhibition of Aurora B, a component of this complex involved in regulating microtubule stability, did not prevent the preferential formation of spindles around kinetochores during MUG (Fig. S3 F). This suggests that another CPC-mediated pathway is responsible.
Intriguingly, unpaired kinetochores detached from the bulk of chromatin can form a spindle even in the absence of centrosomes, albeit with relatively low efficiency. However, our results do not suggest that chromosome arms are irrelevant or the "corpse at a funeral" (Mazia, 1961). The gradient of RanGTP formed by the arms attracts astral microtubules, thus facilitating capture of kinetochores. Although a RanGTP gradient can bias most astral microtubules to grow toward the chromatin, this condition cannot result in the formation of a stable spindle in somatic cells. This is consistent with previous observations that, in the absence of kinetochores, two adjacent centrosomes cannot form a stable spindle structure and ultimately move away from one another (Faruki et al., 2002). However, when kinetochores and centrosomes work together, spindle assembly proceeds even without the RanGTP-mediated activity of the chromatin (Nishitani et al., 1991;Kaláb et al., 2006).
RanGTP-mediated microtubule nucleation and stabilization (Carazo-Salas et al., 1999;Wilde et al., 2001) are undeniably sufficient for the formation of a functional spindle around chromatin-coated beads in Xenopus egg extracts (Heald et al., 1996). However, until now, it has not been possible to determine whether a spindle assembles around "naked" chromatin in somatic cells; kinetochores have always been an integral component of the overall chromosomal structure. Similarly, the fate of


s2fieldsofstudy: Biology Medicine

text:
Comparison of three radiolabelled peptide analogues for CCK-2 receptor scintigraphy in medullary thyroid carcinoma

Purpose Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in a high percentage of medullary thyroid carcinomas (MTC). Analogous to somatostatin receptors, CCK-2 receptors might be viable targets for radionuclide scintigraphy and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed, and some have been carried through into clinical studies. However, these studies are mostly limited and difficult to compare. The aim of this study was to evaluate the diagnostic and therapeutic potential of three promising CCK-2 receptor-binding radiopeptides in patients with MTC. Methods 111In-DOTA-(D)Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2 (111In-DOTA-CCK), a CCK analogue, and the gastrin-based ligands 99mTc-N4-Gly-(D)Glu-(Glu)5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (99mTc-demogastrin 2) and 111In-DOTA-(D)Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (111In-DOTA-MG11) were each administered to the same group of six patients. Planar images made at 3–5, 7 and 24 h p.i. were used for comparison of tumour visualisation and renal uptake. Results 99mTc-demogastrin 2 scintigraphy visualised all known lesions and new lesions in four of six patients. 111In-DOTA-CCK and 111In-DOTA-MG11 on the other hand missed several lesions; tumour uptake of these two radiopharmaceuticals was quite low. Comparison of retention of renal activity showed no major differences between the three radiopeptides. Conclusion 99mTc-demogastrin 2 scintigraphy appeared most promising as a diagnostic tool in patients with MTC. Further studies are required to evaluate its value in patient management. Direct comparisons of the compounds studied strongly suggests that 111In-DOTA-CCK and 111In-DOTA-MG11 have less potential as imaging agents than 99mTc-demogastrin 2. These DOTA-linked compounds are considered unlikely to be useful for radionuclide therapy because of low tumour uptake.

parafollicular cells (C cells) in the thyroid. Elevated serum calcitonin is a very sensitive and specific tumour marker for MTC. It can indicate the presence of remaining or recurring tumour after resection of the primary tumour. However, with the available diagnostic imaging modalities [i.e. computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (US), somatostatin receptor scintigraphy (SRS), 18 F-fluoro-2-deoxyglucose positron emission tomography ( 18 F-FDG PET)] localisation of the recurrent tumour or metastases (and surgical intervention) often is not possible. In cases of distant metastases there is no wellaccepted treatment available. This makes the development of both a sensitive diagnostic imaging modality and a systemic therapy very important.
The high sensitivity of the commonly used pentagastrin stimulation test suggests a widespread expression of cholecystokinin 2/gastrin receptors (CCK-2 receptors) in MTC and indeed autoradiographic studies demonstrated CCK-2 receptors in 90% of MTCs (and in a high percentage of several other tumours like small cell lung cancer) [1]. These G protein-coupled receptors bind both CCK and gastrin and might serve as targets for radiolabelled CCK or gastrin analogues for both scintigraphy and peptide receptor radionuclide therapy (PRRT), analogous to the successful use of radiolabelled somatostatin analogues in patients with tumours overexpressing somatostatin receptors [2,3], Studies in patients with MTC have been performed with both gastrin-like [4][5][6][7] and CCK-like peptides [8]. Unfortunately, comparison of the results of these studies is difficult because of the different protocols applied in noncomparable and sometimes small groups of patients.
The aim of this study was to investigate the biodistribution and compare the diagnostic potential of the three radiolabelled CCK-2 receptor-binding analogues and evaluate the therapeutic potential of the two 111 In-DOTA compounds in the same patients.

Materials and methods
Patients MTC patients with elevated calcitonin levels after thyroidectomy were sent to the Department of Nuclear Medicine for tumour staging with CCK-2 receptor scintigraphy and/ or to investigate the possibility of PRRT with CCK-2 receptor-binding analogues. Patients underwent scintigraphy with 111 In-DOTA-CCK and 99m Tc-demogastrin 2. In patients showing tumour uptake, 111 In-DOTA-MG11 scintigraphy was performed additionally with the aim of localising the maximum possible number of tumour lesions before operation and/or evaluating its potential for radionuclide therapy in patients with distant metastases. Six patients (five women and one man, age: 17-71 years, median: 41 years) were tested with all three mentioned radioligands and included in this study. Calcitonin levels before administration of the first peptide ranged from 0.23 to 131 μg/l (mean: 38.4 μg/l). Use of 111 In-DOTA-CCK and 99m Tc-demogastrin 2 in patients was approved by the Erasmus MC Medical Review Ethics Committee. 111 In-DOTA-MG11 scintigraphy was performed on a compassionate use basis as allowed by the Erasmus MC Medical Review Ethics Committee. All patients gave written informed consent before each administration. The maximal interval between the three scintigrams was 5 weeks in five patients and 4 months in one patient (patient 1).

Peptide handling, radiolabelling and radiochemical analysis
Radiolabelling of DOTA-MG11 (Radiological Chemistry, University Hospital Basel, Switzerland) and DOTA-CCK (BioSynthema, St. Louis, MO, USA) was performed as described earlier [10]. The procedure was optimised to prevent oxidation and radiolysis. Likewise, the procedure of radiolabelling demogastrin 2 (NCSR "Demokritos", Athens, Greece) [7] was slightly modified to achieve higher specific activity while preventing oxidation and radiolysis [11]. Formation and radiochemical purity of the three radiopharmaceuticals were analysed by a combination of instant thin-layer chromatography (ITLC) and highperformance liquid chromatography (HPLC). For all three radiopharmaceuticals an administration of 10 μg peptide with the maximal achievable amount of radioactivity was aimed at, resulting in administered doses as described below.
Imaging was performed with a dual head camera (Prism 2000XP, Philips, Cleveland, OH, USA) equipped with either a medium-energy ( 111 In) or a low-energy highresolution ( 99m Tc) collimator. Dynamic images of the upper abdomen or known tumour lesions were obtained during 30 min after injection. Total body images of 10 min acquisition time were made at 40 min post (start of) injection (p.i.). Static spot views were obtained at 1, 3-5 and 24 h p.i. and also at 7 h p.i. in most cases. Acquisition time of most images was 15 min; only at 24 h p.i. was the scan time for the 99m Tc-labelled compound 30 min. SPECT images of neck and upper abdomen were made at 4-5 h p.i. and also at 24 h p.i. with the 111 In-labelled radioligands.
Planar images of 3-5, 7 and 24 h p.i. were used for comparison of the scintigraphy results.
Interpretation of the images Four nuclear physicians, blinded for the agents used and the locations of the known lesions, evaluated the scans conjointly. Lesions that did not represent normal biodistribution and persisted over time were judged as suspicious for tumour localisation ("positive"). Lesions that were barely visible and/or did not show persisting uptake over time were judged as equivocal. After the random analysis of scans per ligand, also a combination of series of the three ligands was compared with regard to biodistribution and pathological uptake.
Scintigraphic findings were correlated with diagnostic investigations that had been performed earlier. In cases of equivocal or positive lesions in regions without known tumour lesions, correlating imaging was performed. For neck lesions US investigations were performed by an experienced radiologist (AvdL) with knowledge of the location of the lesions on the CCK-2 receptor scintigrams. If corresponding lymph nodes could be localised, fine-needle aspiration cytology was performed (if technically possible). For other unknown lesions CT and/or MRI were performed which were interpreted by the radiologist with knowledge of the affected organ using standard radiological criteria for malignancy like enhancement patterns, invasiveness, bone destruction and density (CT) or signal intensity (MRI) of lesions. Thereafter, the CT and MR images were correlated with the CCK-2 receptor scintigrams.
Renal uptake was calculated according to the geometric mean method using anterior and posterior planar views and a standard with known activity. Data were corrected for background activity and attenuation (based on CT data). In each patient the same region was used for all calculations (both time points of the three radioligands). If overprojection of activity in other organs hampered the measurement, total counts in the kidney were estimated by multiplying the number of counts/pixel (in the part of the kidney without overprojection) with the number of pixels of the kidney (as defined on early images without overprojection).

Analysis of blood and urine
Radioactivity in blood samples was measured with a Packard COBRA auto-gamma counting system (Packard, Meriden, CT, USA) and radioactivity in urine samples with a dose calibrator. Blood samples were drawn at 10, 20, 30, 40, 60 and 90 min and 2, 4 and 24 h after start of injection. Urine was collected during 24 h after injection. The chemical status of the radioactive compounds in blood and urine was analysed as a function of time using HPLC techniques.
Calcitonin levels were determined by the standard laboratory procedure in the hospital (radioimmunoassay, upper normal value 0.14 μg/l) in blood samples taken shortly before and at 2, 5 and 10 min after start of administration of the radioligands.

Administration
Administration of the radioligands was often followed by a clear increase of serum calcitonin levels ( Fig. 1) and was sometimes associated with mild side effects; these were different in the various patients. Side effects during administration were increase of heart rate (maximum heart rate 105 bpm, rise only experienced by one patient), flushes (four patients), mild pressure on the chest (two patients), mild nausea (four patients, no vomiting), paraesthesia in the hands (two patients) and in one patient some dizziness. In one patient (patient 5) 111 In-DOTA-CCK caused the most side effects; this was the only patient with calcitonin levels clearly higher after administration of 111 In-DOTA-CCK compared to calcitonin levels after administration of the other radioligands (Fig. 1). In three (of six) patients, 99m Tc-demogastrin 2 caused the most side effects and two patients did not have any side effects at all. All side effects were mild and disappeared spontaneously within 10 min after administration (heart rate normalised within 2 min). Two patients previously had a pentagastrin stimulation test and both judged the side effects of that test to be far more severe.

Biodistribution
The dynamic series obtained during the first 30 min p.i. showed radioactivity in blood pool, kidneys and stomach in all patients. Visualisation of the (strong CCK-2 receptorpositive [12]) stomach was clearest with 99m Tc-demogastrin 2. Total body images showed accumulation in joint regions (such as shoulders, hips, lumbar spine), nipples and in breasts of premenopausal women as well. In some patients tumour uptake was already visible in this early time frame. On the 3-4 h p.i. images, clear visualisation of the stomach was observed, especially with 99m Tc-demogastrin 2. Besides uptake in joint regions, breasts and nipples and the activity in the kidneys and bladder, there was also radioactivity in the bowel. 111 In-DOTA-CCK showed some activity in the liver, 111 In-DOTA-MG11 showed less liver activity and with 99m Tc-demogastrin 2 the liver was hardly visible. At this time point more tumour lesions were recognised, best with 99m Tc-demogastrin 2. Physiological activity in joint regions and breasts decreased over time, whereas activity in tumour lesions was more stable. This resulted in easier interpretation of the images at later time points, especially at 7 h p.i. (Fig. 2). At 24 h p.i. most tumour lesions were still visible, even with 99m Tc-demogastrin 2, partly due to the very low background activity.
Lesion detection 99m Tc-demogastrin 2 visualised all known lesions. In addition this analogue visualised in four patients unknown lesions in the neck (two patients), brain (one patient), bone (one patient) and/or liver (one patient). With the other two compounds several known lesions were missed and fewer new lesions were discovered. New liver lesions in one patient were not visible with 111 In-DOTA-CCK; neck lesions in another patient could only be recognised on the 99m Tc-demogastrin 2 scan. An unknown lesion in the brain was only retrospectively seen with the two 111 In-DOTA compounds (after localisation with 99m Tc-demogastrin 2). In each of the four patients with new lesions at least one of the new lesions was confirmed by additional imaging with US, CT or MRI. A neck lesion only visible on 99m Tcdemogastrin 2 scintigraphy was confirmed by US-guided fine-needle aspiration cytology as a 6-mm true positive lesion (patient 4). In patient 2 cytologic investigation of the brain lesion was not possible because of the location of the lesion. MRI images could not differentiate between tumour or pineal cyst. Results are listed in Table 1. Figures 3 and 4 show examples of the comparative images of the three radiopharmaceuticals in two of the six patients studied.

Renal uptake
Visually there were no clear differences in renal uptake between the three compounds; the stomach (CCK-2 receptor-positive) to kidney ratio seemed best with 99m Tcdemogastrin 2 (Fig. 5). Renal uptake calculation was hampered by overprojection of activity in stomach and bowel. To recognise important differences in renal uptake, regions without overprojection of other activity-accumulating organs were chosen. In three patients these regions were available for all three radioligands (and time points), and in these patients renal uptake values were in the same range (uptake in left kidney: for 111 In-DOTA-CCK, 111 In-DOTA-MG11 and 99m Tc-demogastrin 2, respectively, 0.62±0.27, 0.37±0.19 and 0.48±0.22%ID at ±4 h p.i. and 0.25±0.10, 0.16±0.04 and 0.27±0.07%ID at ±24 h p.i.).

Radioactivity in blood and urine
Serum radioactivity and urine radioactivity of all three radioligands were analysed in five patients; detailed results are described elsewhere [11]. Briefly, there were neither statistically significant differences in the decrease of serum radioactivity nor in the (very fast) urinary excretion of radioactivity for the three compounds. HPLC blood analyses, however, showed clear differences in the degradation rates of the analogues. In blood samples taken at 10 min after start of administration 64±6% of 99m Tc-demogastrin 2 was still intact. At the same early time point only 38±9% of 111 In-DOTA-CCK and 9±3% of 111 In-DOTA-MG11 was still intact.

Discussion
Patients with residual or recurrent MTC after surgery can be potentially cured if lesions are limited to the neck region. Inasmuch as available diagnostic modalities often fail to detect the tumour lesions in patients with slightly elevated serum calcitonin levels, a more sensitive modality is needed. Furthermore, there is a strong impetus for the development of PRRT, based on targeting CCK-2 receptors, because there is currently no effective therapy available for patients with distant metastases of MTC.
Comparison of the results of several studied CCK-2 receptor-binding compounds [4][5][6][7][8] used in various and sometimes small patient groups is of limited value. Nevertheless, the promising results of a number of preclinical, as well as a few clinical studies using radiolabelled CCK-2 receptor-binding peptides, have shown that these compounds might be useful in the management of patients with MTC (and possibly other CCK-2 receptorpositive tumours [1,13]). We therefore carried out a more controlled study that allows for the direct comparison of promising CCK-2 receptor-targeted analogues in the same patients in an attempt to find the best analogue for tumour localisation and test the potential of the two 111 In-DOTA compounds for PRRT.
Comparing the results of the analogues tested, 99m Tcdemogastrin 2 scintigraphy showed the most promising results. All known tumour lesions were visualised and in four of six patients new lesions were discovered. Even in two patients with rather low calcitonin levels (patients 3 and 4) lesions were visible. Both 111 In-DOTA-MG11 and 111 In-DOTA-CCK were less suitable for scintigraphy as image quality was inferior and several lesions were missed that were observed with 99m Tc-demogastrin 2. The scintigraphic results of the 111 In-labelled peptides were disappointing as not only the sensitivity but also the uptake in the visible lesions was limited and appeared to be insufficient for radionuclide therapy in these tumours, especially considering that MTC is reported to be radioresistant [14].
The variation in diagnostic performance among the three tracers is probably the combined result of several parameters. It is well known that 99m Tc has better imaging properties than 111 In. Furthermore, a higher specific activity labelling could be achieved with 99m Tc-demogastrin 2 resulting in a higher injected activity (labelled to 10 μg of the peptide). Another important parameter probably is the difference in the in vivo stability of the three radioligands. The stability of 111 In-DOTA-MG11 was unexpectedly low. This peptide was previously reported to be stable in ex vivo human serum [9], but HPLC results of blood samples taken at 10 min after start of the administration showed that only approximately 10% of the original peptide was still intact. In contrast, 99m Tc-demogastrin 2 was more stable; HPLC showed that on average more than 60% of the radioligand was intact at the same time point. Recently Good et al. reported a decreased mean lifetime and lower tumour uptake of 111 In-DOTA-MG11 in comparison with 111 In-DTPA-DGlu 1 -minigastrin [15]. Therefore, the diagnostic efficacy of 111 In-DOTA-MG11 might be inferior to that of 111 In-DTPA-DGlu 1 -minigastrin [5,6]. Gotthardt et al. reported a tumour detection rate of 87% by scintigraphy using this compound in a group of 26 MTC patients. Most of the patients included in that study, however, had known metastases and in the group of patients with occult disease (n=5) tumour lesions were found in only one patient. A direct comparison between 111 In-DTPA-DGlu 1minigastrin and 99m Tc-demogastrin 2 would be of interest.
The renal uptake of the three test compounds was also of interest, considering the previous report by Béhé and Behr [5], who observed that therapy with 90 Y-DTPA-DGlu 1minigastrin caused renal damage. Calculation of renal uptake values was impaired by overprojection of activity in bowel and stomach that changed over time. However, visual evaluation and an estimation of renal uptake made clear that the differences in renal uptake were far less than expected. In particular, the renal uptake of 99m Tc-demogastrin 2 was in the same range as the uptake of 111 In-DOTA-MG11 and 111 In-DOTA-CCK, although in previous animal studies kidney radioactivity levels were high in comparison with 111 In-DOTA-MG11 and 111 In-DOTA-CCK [7,9,16]. Béhé et al. [17] reported high uptake of 111 In-DTPA-DGlu 1 -minigastrin in the kidneys of mice. This renal uptake could be significantly blocked by coinjection of polygluta- In-DOTA-CCK at 4, 7 and 24 h p.i., respectively (activity in breast tissue decreasing over time). Clearest visualisation of known tumour lesions in neck, lungs, liver and bone with 99m Tc-demogastrin 2 (some lesions marked with arrows) mic acids, whereas tumour uptake was not impaired, resulting in a higher tumour to kidney ratio. If polyglutamic acid coinjection is well tolerated by patients, this method might be useful to reduce nephrotoxicity after PRRT with 90 Y-DTPA-DGlu 1 -minigastrin or other gastrin-like analogues. The commonly used plasma expander Gelofusin® might also be useful for lowering renal uptake. Gotthardt et al. reported a significant reduction of renal accumulation (45%) of 111 In-DTPA-DGlu 1 -minigastrin in rats using Gelofusin® [18]. Potential bone marrow toxicity of 90 Y-DTPA-DGlu 1 -minigastrin might remain a cause of concern, because the reported activity in blood and other organs was not lower after use of polyglutamic acids [16]. These various findings imply that further improvement can be made to enhance the efficacy of CCK-2 receptor-binding ligands for PRRT. As 99m Tcdemogastrin 2 showed most tumour lesions and renal uptake of this analogue was lower than expected, 188 Re-demogastrin 2 might be one of the potential radiopharmaceuticals which deserves further investigation.
From our results we conclude that: (1) 99m Tc-demogastrin 2 scintigraphy appears to be a very promising diagnostic tool in patients with evidence of recurrence or metastases of MTC. Further studies are warranted to investigate its value in patient management. (2) In this small group of patients tumour uptake of either 111 In-DOTA-MG11 or 111 In-DOTA-CCK was less promising for imaging and was considered too low to expect any beneficial effect from PRRT. To enable therapy with CCK-2 receptor-binding radiolabelled analogues further research is needed and should be aimed towards designing stable compounds which retain high receptor affinity but in addition have more favourable biodistribution profiles including low renal retention.