Apoptosis is associated with triacylglycerol accumulation in Jurkat T-cells.

Magnetic resonance spectroscopy is increasingly used as a non-invasive method to investigate apoptosis. Apoptosis was induced in Jurkat T-cells by Fas mAb. (1)H magnetic resonance spectra of live cells showed an increase in methylene signal as well as methylene/methyl ratio of fatty acid side chains at 5 and 24 h following induction of apoptosis. To explain this observation, (1)H magnetic resonance spectra of cell extracts were investigated. These demonstrated a 70.0+/-7.0%, 114.0+/-8.0% and 90.0+/-5.0% increase in the concentration of triacylglycerols following 3, 5 and 7 h of Fas mAb treatment (P<0.05). Confocal microscopy images of cells stained with the lipophilic dye Nile Red demonstrated the presence of lipid droplets in the cell cytoplasm. Quantification of the stained lipids by flow cytometry showed a good correlation with the magnetic resonance results (P > or =0.05 at 3, 5 and 7 h). (31)P magnetic resonance spectra showed a drop in phosphatidylcholine content of apoptosing cells, indicating that alteration in phosphatidylcholine metabolism could be the source of triacylglycerol accumulation during apoptosis. In summary, apoptosis is associated with an early accumulation of mobile triacylglycerols mostly in the form of cytoplasmic lipid droplets. This is reflected in an increase in the methylene/methyl ratio which could be detected by magnetic resonance spectroscopy.

Unique case of fatal carbon monoxide poisoning in the absence of a combustible fossil fuel.

A 37-year-old man died as a result of exposure to carbon monoxide within an apartment. An investigation of the apartment showed no gas appliances or gas supply to the apartment and no evidence of any combustion event to any part of the apartment or roof space. Inhalation of dichloromethane was excluded. Heating to the apartment was found to be via an electrical storage heater, the examination of which revealed that the cast-iron core and insulating material showed evidence of heat damage with significant areas devoid of carbon. This electric storage heater is hypothesized to be the source of carbon for the fatal production of carbon monoxide within the apartment.

Treatment of SW620 cells with Tomudex and oxaliplatin induces changes in 2-deoxy-D-glucose incorporation associated with modifications in glucose transport.

UNLABELLED: Many studies suggest that changes in the uptake of the glucose analog FDG after therapy, compared with pretreatment uptake, predicts tumor response to therapy. However, clinical interpretation is compromised by a limited understanding of the effect of therapy on FDG and 2-deoxy-D-glucose (DG) uptake at the tumor cell level. METHODS: Uptake of 2-deoxy-D-[1-(3)H]glucose (3H-DG) by SW620 colonic tumor cells was measured before and 8, 16, 24, and 48 h after treatment with the novel platinum drug oxaliplatin and the novel thymidylate synthase inhibitor Tomudex. Glucose transport was determined by measuring the initial rate of uptake of the nearly nonmetabolized glucose analog 3-O-methyl-D-[1-(3)H]glucose (3H-OMG). The effect of these drugs on cell cycle kinetics was determined using flow cytometry. RESULTS: Treatment of SW620 cells with oxaliplatin was found to decrease uptake of 3H-DG after up to 24 h, but uptake returned to control levels after longer treatment. The initial decrease in 3H-DG incorporation was associated with a lower rate of glucose transport. Treatment of cells with Tomudex induced an increase in 3H-DG uptake that depended on treatment duration. Both glucose transport and the volume of distribution of 3H-OMG were higher in Tomudex-treated cells than in control cells. Flow cytometry showed that oxaliplatin induced a G2 and M arrest, whereas a buildup of cells in the S phase was associated with Tomudex treatment. Both treatments induced apoptosis in SW620 cells. CONCLUSION: Changes in uptake of DG by SW620 colonic tumor cells responding to therapy is specific to the drug type. Modulation of glucose transport was associated with changes in 3H-DG uptake.

Comparison of thymidylate synthase (TS) protein up-regulation after exposure to TS inhibitors in normal and tumor cell lines and tissues.

Thymidylate synthase (TS) is an important target for cancer chemotherapy. However, several mechanisms of resistance to TS inhibitors have been described. One mechanism that may be relevant to short-term exposure to TS inhibitors occurs as a result of disruption of the autoregulatory loop, which allows TS to control its own translation. This disruption leads to up-regulation of TS protein and is generally thought to decrease efficacy. This study has investigated TS protein up-regulation using a range of TS inhibitors in both tumor and nonmalignant cell lines in vitro and in vivo. Up-regulation of TS protein showed a time-, dose-, and cell-type-specific response to treatment with ZD9331. This response was observed in W1L2 cells treated for 24 h at equitoxic doses of raltitrexed (6-fold), ZD9331 (10-fold), fluorouracil (5-fold), LY231514 (7-fold), AG337 (7-fold), and BW1843U89 (3-fold). Up-regulation was observed over a range of doses. Elevation of TS protein only persisted up to 12 h after removal of drug. The extent of induction does not depend on basal TS levels. Nontransformed human fibroblasts showed significantly greater up-regulation of TS protein than tumor cells exposed to an equitoxic dose of ZD9331. In vivo experiments using the L5178Y thymidine kinase -/- mouse lymphoma implanted into DBA2 mice also showed greater up-regulation of TS protein in normal intestinal epithelial cells compared with tumor cells. These results confirm that TS up-regulation is a common feature of TS inhibition in tumor cells and that it may occur to a greater extent in normal tissues, although the clinical implications of these findings remain to be determined.

Deoxyglucose uptake by a head and neck squamous carcinoma: influence of changes in proliferative fraction.

PURPOSE: Positron emission tomography, using the glucose analogue fluorodeoxy-D-glucose (FDG), is proving to be useful in the early response detection of head and neck tumors. Presently mechanisms underlying changes in FDG uptake after therapy are poorly understood. Response of tumors to therapy is often accompanied by a decrease in tumor cell proliferation. The purpose of this study was to assess whether or not changes in the uptake of deoxyglucose (DG) may reflect differences in proliferative fraction independent of other metabolic changes induced by using therapeutic agents. METHODS AND MATERIALS: HN5 head and neck tumor cells were grown to different cell densities producing populations of cells with different proliferative indices without the need for exogenous agents to manipulate cell-cycle kinetics. (3)H-DG uptake, S-phase fraction (Spf), and lactate production were determined in each population of cells. RESULTS: Large differences in Spf between populations of cells were associated with differences in DG incorporation. Lactate production was also found to correlate strongly with DG uptake. CONCLUSION: Therapy-induced changes in FDG uptake by tumors may be partly due to changes in proliferation.

Effect of p53 status on sensitivity to platinum complexes in a human ovarian cancer cell line.

Wild-type p53 is frequently mutated in late-stage ovarian cancer and has been proposed as a determinant of cisplatin chemosensitivity. We have therefore established a human ovarian cancer cell line differing only in p53 status and characterized its response after treatment with different platinum complexes. The wild-type p53-expressing cell line A2780 was stably transfected with HPV-16 E6 (E6) or an empty vector (VC) as control. Parental A2780 and VC had similar cisplatin sensitivities, whereas E6 was 3- to 4-fold more sensitive as measured by sulforhodamine B and clonogenic assay. E6 was 2- to 3-fold more sensitive to transplatin and the novel cisplatin analog ZD0473 than VC, whereas the trans-platinum analog JM335 was approximately equitoxic. Platinum uptake was similar for all of the cell lines after cisplatin. The removal of platinum-DNA adducts, as measured by atomic absorption spectroscopy, was reduced in E6 compared with VC after cisplatin but similar after JM335. After 10 microM cisplatin, the G(1) population (0-96 h) was reduced in E6 cells compared with VC, whereas the S phase (8-48 h) and G(2) phase (48-96 h) were increased. Similar proportions of VC and E6 cells died by apoptosis, as detected by annexin V binding, but more E6 cells died by necrosis relative to VC. Our results suggest that the loss of functional p53 can increase cisplatin cytotoxicity in A2780, with loss of G(1)/S checkpoint control and decreased cisplatin-DNA adduct repair, but these effects can be circumvented by the use of JM335, which forms different DNA-platinum adducts.

Differentiation of separated mouse mammary luminal epithelial and myoepithelial cells cultured on EHS matrix analyzed by indirect immunofluorescence of cytoskeletal antigens.

We have previously demonstrated that purified virgin mouse mammary luminal epithelial and myoepithelial cells promiscuously express cell type-specific cytokeratins when they are cloned in vitro. Changes in cytokeratin expression may be indicators of the loss or change of the differentiated identity of a cell. To investigate the factors that may be responsible for the maintenance of differentiated cellular identity, specifically cell-cell and cell-matrix interactions, we cloned flow-sorted mouse mammary epithelial cells on the extracellular matrix (ECM) derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Changes in cell differentiation on EHS, compared with culture on glass, were analyzed by comparing patterns of cytokeratin expression. The results indicate that ECM is responsible for maintenance of the differentiated identity of basal/myoepithelial cells and prevents the inappropriate expression of luminal antigens seen on glass or plastic. Luminal cell identity in the form of retention of luminal markers and absence of basal/myoepithelial antigens, on the contrary, appears to depend on homotypic cell-cell contacts and interactions. The results also show that luminal cells (or a subpopulation of them) can generate a cell layer that expresses only basal cytokeratin markers (and no luminal cytokeratin markers) and may form a pluripotent compartment. (J Histochem Cytochem 47:1513-1524, 1999)

Lack of prognostic significance of ploidy and S-phase measurements in advanced ovarian cancer.

DNA ploidy and S-phase measurements were made in fresh tumour samples obtained from 27 patients with either untreated or previously treated ovarian cancer. In addition, an assessment of in vitro chemosensitivity to post-operative chemotherapy was carried out. Whilst we found that patients with aneuploid tumours tended to survive longer (median survival 441 days versus 268 days for diploid tumours; p = 0.308) and this was associated with clinical response (p = 0.137) this was not statistically significant. Tumours with a high S-phase fraction (i.e. > 9% median) showed a median survival of 362 days compared with 484 days for those with a low S-phase fraction (< 9%; p = 0.79). Whilst a high level of predictability for chemosensitive patients (81%) was achieved, chemosensitivity per se was not related to the respective tumour DNA ploidy or S-phase fraction. In addition, when considering disease stage i.e. FIGO stage III or IV, or extent of residual disease between diploid and aneuploid groups, there were no statistically significant differences. However, there was a tendency for stage III tumours to be aneuploid (p = 0.189). Aneuploid tumours also showed the highest S-phase fractions (p = 0.03), indicative of a high proliferation rate. In conclusion, we have shown that DNA content and ploidy status do not appear to be major prognostic indicators for the management of ovarian cancer.

Uptake of glucose analogues by colonic tumour cells during growth and after treatment with hydroxyurea.

SW620 cells were grown in tissue culture flasks to various cell densities producing populations of cells with a range of proliferative indices. The uptake of the two glucose analogues, deoxy-D-glucose (DG) and 3-O-methylglucose (OMG) was determined and found to be associated with S-phase fraction. The strong correlation between DG and OMG uptakes suggested that proliferation-related changes in transmembrane transport accounted for the association with S-phase fraction. Treatment of SW620 cells with the cell cycle inhibitor hydroxyurea was found to increase the uptake of DG and OMG in a time-dependent manner.

Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis.

Apoptosis was induced by treating L1210 leukaemia cells with mechlorethamine, and SW620 colorectal cells with doxorubicin. The onset and progression of apoptosis were monitored by assessing caspase activation, mitochondrial transmembrane potential, phosphatidylserine externalization, DNA fragmentation and cell morphology. In parallel, 31P magnetic resonance (MR) spectra of cell extracts were recorded. In L1210 cells, caspase activation was detected at 4 h. By 3 h, the MR spectra showed a steady decrease in NTP and NAD, and a significant build-up of fructose 1,6-bisphosphate (F-1,6-P) dihydroxyacetonephosphate and glycerol-3-phosphate, indicating modulation of glycolysis. Treatment with iodoacetate also induced a build-up of F-1,6-P, while preincubation with two poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide and nicotinamide, prevented the drop in NAD and the build-up of glycolytic intermediates. This suggested that our results were due to inhibition of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Doxorubicin treatment of the adherent SW620 cells caused cells committed to apoptosis to detach. F-1,6-P was observed in detached cells, but not in treated cells that remained attached. This indicated that our observations were not cell line- or treatment-specific, but were correlated with the appearance of apoptotic cells following drug treatment. The 31P MR spectrum of tumours responding to chemotherapy could be modulated by similar effects.

Quantitative changes in cytological molecular markers during primary medical treatment of breast cancer: a pilot study.

AIM: To quantify the changes in biological molecular markers during primary medical treatment in patients with operable breast cancer and to assess their possible relationship with response to treatment. METHODS: The treatment group consisted of 31 patients with operable breast carcinomas, median age 57 years (range 41-67), treated with four 3-weekly cycles of chemotherapy with Mitoxantrone, methotrexate (+/- mitomycin C), and tamoxifen before surgery. Fine needle aspiration (FNA) was used to obtain samples from patients prior to and at 10 or 21 days post-treatment. The following molecular markers were assessed: estrogen receptor (ER), progesterone receptor (PgR), p53, Bcl-2, and Ki67 measured by immunocytochemistry, and ploidy and S-phase fraction (SPF) by flow cytometry. To evaluate the reproducibility of the technique, repeat FNA was performed in a separate non-treatment control group of 20 patients and the same molecular markers assessed, two weeks after the first sample with no intervening treatment. RESULTS: The non-treatment control group showed a high reproducibility for the measurement of molecular markers from repeat FNA. In the treatment group there was a non-significant reduction in SPF and a significant reduction (p = 0.005) in Ki67. Patients who responded to neoadjuvant therapy were more likely to have a reduction in these two markers than those who failed to respond. Similarly, a reduction in ER scores was observed between the first and second samples (p = 0.04). For PgR, the change between the first and second samples was not significant although there was a significant difference between responders and non-responders (p = 0.03). All nine patients with an increase in PgR were responders. No significant changes in p53 or Bcl-2 were observed during treatment. CONCLUSION: Molecular markers can be adequately measured from FNA samples prior to and during neoadjuvant therapy. Changes in cellular proliferation and hormone receptors have been shown that may be related to tumour response. These relationships should be assessed in a larger cohort of patients.

A novel class of lipophilic quinazoline-based folic acid analogues: cytotoxic agents with a folate-independent locus.

Three lipophilic quinazoline-based aminomethyl pyridine compounds, which differ only in the position of the nitrogen in their pyridine ring, are described. CB300179 (2-pyridine), CB300189 (4-pyridine) and CB30865 (3-pyridine) all inhibited isolated mammalian TS with IC50 values of 508, 250 and 156 nM respectively. CB30865 was the most potent growth inhibitory agent (IC50 values in the range 1-100 nM for several mouse and human cell types). CB300179 and CB300189 were active in the micromolar range. Against W1L2 cells, CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. Furthermore, combinations of precursors and end products of folate metabolism, e.g. dThd/hypoxanthine (HX) or leucovorin (LV), did not prevent activity. CB30865 did not interfere with the incorporation of tritiated dThd, uridine or leucine after 4 h. A cell line was raised with acquired resistance to CB30865 (W1L2:R865; > 200-fold), which was not cross-resistant to CB300179 or CB300189. In addition, W1L2:R865 cells were as sensitive as parental cells to agents from all the major chemotherapeutic drug classes. CB300179 and CB300189 induced an S phase accumulation (preventable by co-administration of dThd). No cell cycle redistribution was observed following exposure (4-48 h) to an equitoxic concentration of CB30865. In the NCI anticancer drug-discovery screen, CB30865 displayed a pattern of activity which was not consistent with known anti-tumour agents. These data suggest that CB30865 represents a class of potent potential anti-tumour agents with a novel mechanism of action.

Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting.

Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as clones. The separation of virgin mouse mammary luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting, their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1 cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types, two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression of differentiation-specific cytoskeletal markers in vitro.

Cell cycle effects of CB30865, a lipophilic quinazoline-based analogue of the antifolate thymidylate synthase inhibitor ICI 198583 with an undefined mechanism of action.

CB30865 (p-[N-(7-bromo-3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl+ ++)-N-(prop-2-ynyl)amino]-N-(3-pyridylmethyl)benzamide) is a quinazoline-based pyridine-containing compound that emerged from a programme aimed at the development of thymidylate synthase (TS) inhibitors as anticancer agents. Its structure is based on the antifolate ICI 198583, but with a pyridine ring replacing the glutamate. Despite its structure, CB30865 does not act in vitro via inhibition of TS or, apparently, other known folate-dependent pathways, and extensive mechanistic studies suggest that it acts via a novel locus with respect to conventional antitumour agents. However, CB30865 is highly potent against a variety of human tumour cell lines (e.g., 50%-inhibitory concentration [IC50] values in the 1-10 nM range). Thus, the cell cycle effects of CB30865 were investigated. DNA histogram analysis of W1L2 human lymphoblastoid, L1210 murine leukaemia, and CH1 human ovarian cells (propidium iodide staining) has demonstrated that CB30865 does not cause a phase-specific arrest at concentrations that have been shown to inhibit colony formation. This is unexpected for an anticancer agent. In W1L2 cells, using bromodeoxyuridine (BrdUrd) labelling and bivariate Hoechst/ propidium iodide staining, it was revealed that 0.003-0.15 microM CB30865 (1-50 x 72 h IC50) caused cells to arrest in all phases of the cell cycle simultaneously after 20-24 h exposure. This effect was also observed in CH1 and L1210 cells, though the arrest was at slightly different times. Thus, using this technique, it has been demonstrated that CB30865 induces an unusual and delayed cell cycle arrest, which provides further evidence for a novel locus of action for this compound.

Analysis and sorting of apoptotic cells from fine-needle aspirates of excised human primary breast carcinomas.

Numerous recent studies have indicated the central role of apoptosis as a determinant of the growth abnormalities occurring with malignancy and of the effectiveness of a wide range of therapeutic manoeuvres in cancer treatment. However, there has been a relative paucity of studies measuring apoptosis in human solid tumours, because of the low incidence of apoptotic cells, the difficulty of identifying cells undergoing apoptosis, and the ethical and practical restrictions on obtaining repeat biopsies from patients during therapy. Fine-needle aspirates (FNAs) may be obtained from breast carcinomas as a minimally invasive technique allowing repeat sampling. We describe an approach in which the in situ end labelling (TUNEL) assay is applied to cells in FNAs prior to their analysis by flow cytometry, which allows many thousands of cells to be analysed automatically by objective criteria. Cells that were discriminated as apoptotic on flow cytometric analysis were sorted onto microscope slides and found to show nuclear morphology typical of apoptotic cells. A statistically significant relationship was found between the flow cytometric analysis and the conventional application of TUNEL on histological sections (P = 0.03). Repeat analyses of FNAs from 12 carcinomas showed a median 2.05% apoptotic cells and an overall coefficient of variation of 34.9%. Of the total variability in 12 tumours, 80% was attributed to variation between tumours, 12% between batches, and 8% was random. Thus, this technique should be able to detect the major differences in the percentage of apoptotic cells that occur between different tumours (range 0.3-11.3% by flow cytometry) and between different phases of treatment, and should provide a useful tool for further research on this process in solid tumours.

Characterisation of the p53 status, BCL-2 expression and radiation and platinum drug sensitivity of a panel of human ovarian cancer cell lines.

The P53 gene is frequently mutated in late stage ovarian cancer and has been proposed as a determinant of radiation and chemosensitivity. We have therefore determined the p53 functional status, P53 sequence, radiation sensitivity and cytotoxicity of cisplatin and the novel platinum analogue, AMD473, in a panel of 6 human ovarian cancer cell lines. Constitutive p53 protein levels were low in A2780, CH1, LK1, LK2 and PA1 but were markedly induced following irradiation. In OV1P, constitutive p53 protein was readily detectable and levels were induced slightly following irradiation. p21WAF1/CIP1 and MDM-2 mRNA were constitutively expressed in all the cell lines and expression was induced markedly following irradiation. There was marked radiation induced G1/S arrest in A2780 but only partial arrests in CH1, LK1, LK2, PA1 and OV1P lines. No mutations were found in A2780, CH1, LK1, LK2 and PA1 cells by single-strand conformational polymorphism (SSCP) analysis but a heterozygous point mutation was found in exon 5 of OV1P. All the cell lines were radiation sensitive and also relatively sensitive to cisplatin; however, OV1P was the most resistant being consistent with its heterozygous P53 status. AMD473 was less potent than cisplatin but a similar pattern of drug sensitivity was observed with the exception of LK2, which was resistant. CH1, LK1, LK2 and PA1 all expressed BCL-2 protein but there was no expression in A2780 and OV1P. Our results suggest an overall association between wild type P53 and radiation and platinum drug sensitivity in these ovarian cancer cell lines.

Proliferation is associated with 2-deoxy-D-[1-3H]glucose uptake by T47D breast tumour and SW480 and SW620 colonic tumour cells.

The interpretation of therapy-induced changes in the uptake of fluorodeoxyglucose by tumours, detected using PET, is dependent on which tumour characteristics are associated with its uptake. In this study the relationship between proliferation (S-phase fraction) and the uptake of 2-deoxy-D-[1-3H]glucose by T47D breast tumour and SW480 and SW620 colonic tumour cells was measured between 2 and 12 days after seeding. Strong correlations (p < 0.001) were observed between viable cell number and the uptake of 2-deoxy-D-[1-3H]glucose/flask by each of the cell lines. Uptake of this compound was also found to correlate with S phase fraction in the T47D line (p < 0.05) and the SW480 (p < 0.01) and the SW620 (p < 0.001) colonic tumour lines. The findings of the present study suggest that therapy-induced changes in the uptake of this compound may at least partially reflect changes in proliferative fraction.

Changes in hormone receptors and proliferation markers in tamoxifen treated breast cancer patients and the relationship with response.

AIM: To determine the effects of tamoxifen on the levels of hormone receptors and proliferation markers in the early phase of treatment and the relationship of the changes with tumor response in patients with primary breast cancer. METHODS: Twenty-one women with primary, operable breast carcinomas were treated with tamoxifen 20 mg daily. Fine needle aspiration (FNA) was used to obtain samples prior to the start and at 14 days and 8-weeks post-treatment. From these samples estrogen receptor (ER), progesterone receptor (PgR), and Ki67 levels were determined using immunocytochemistry and ploidy and S-phase fraction (SPF) using flow cytometry. Tumor response was measured clinically according to UICC criteria. RESULTS: There were 12 responders (2 CR, 10 PR) and 9 non-responders (2 NC, 7 PD). Responders were more likely to be ER+ (p = 0.002), PgR+ (p = 0.006), and low SPF (p = 0.06). At 14 days post-tamoxifen, the median decrease in Ki67 (% cells staining) for responders was -4.8 and for non-responders -0.15 (p = 0.005). This decrease was seen predominantly in ER+ tumours. The difference in SPF was not significant. A decrease in ER was seen in 3/15 patients all of whom were responders. A rise in PgR was seen in 7/17 patients and all but one were responders. Similar changes for ER and PgR were seen at 8-weeks post-tamoxifen, although the reductions in Ki67 and SPF at that time point were not related to response. CONCLUSION: We have observed a decrease in Ki67 and ER and a rise in PgR after 14 days of treatment with tamoxifen that was related to subsequent response. This is the first study in which an early decrease in a proliferation marker has been shown to relate to subsequent clinical response.

Functional consequences of cyclin D1 overexpression in human mammary luminal epithelial cells.

The proliferation of eukaryotic cells is primarily regulated by a decision made during the G1 phase of the cell cycle as to remain in the cycle and divide, or to withdraw from the cycle and adopt a different cell fate. During this time, environmental signals, which regulate the synthesis of the G1 cyclins, are coupled to cell division. In this context, mammalian D-type cyclins have been shown to control progression through the G1 phase of the mammalian cell cycle. Specifically, cyclin D1 has been reported frequently to be amplified, over-transcribed and overexpressed in human breast carcinomas. Although the effects of cyclin D1 overexpression have been examined in human breast carcinoma cell lines, the biological consequences of cyclin D1 expression in normal human mammary epithelial cells remain to be elucidated. In this study we have stably over expressed cyclin D1 in human mammary luminal epithelial cells in order to more directly address the role of cyclin D1 in cell cycle control and tumorigenesis of the human breast. Here, we demonstrate that the effect of cyclin D1 overexpression in these cells is to reduce their growth factor dependency, as well as shorten the duration of G1 and correspondingly reduce the mean generation time. Collectively, our data indicate that deregulation of cyclin D1 expression in human mammary epithelial cells can provide a growth advantage and hence contribute to the oncogenic potential of these cells.