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1.
Br J Cancer ; 86(6): 963-70, 2002 Mar 18.
Article in English | MEDLINE | ID: mdl-11953830

ABSTRACT

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.


Subject(s)
Apoptosis , Triglycerides/metabolism , Flow Cytometry , Humans , Jurkat Cells , Magnetic Resonance Spectroscopy , fas Receptor/physiology
2.
J Nucl Med ; 41(10): 1753-9, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11038008

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Organoplatinum Compounds/pharmacology , Quinazolines/pharmacology , Thiophenes/pharmacology , Biological Transport , Cell Cycle/drug effects , Colonic Neoplasms/pathology , Deoxyglucose/metabolism , Flow Cytometry , Glucose/metabolism , Humans , Oxaliplatin , Thymidylate Synthase/antagonists & inhibitors , Time Factors , Tritium , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism
3.
Mol Pharmacol ; 57(3): 503-11, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10692490

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Ovarian Neoplasms/drug therapy , Platinum Compounds/pharmacology , Tumor Suppressor Protein p53/metabolism , Apoptosis/drug effects , Cell Cycle/drug effects , Cisplatin/pharmacology , DNA Adducts , DNA Repair/drug effects , Drug Screening Assays, Antitumor , Female , Humans , Organoplatinum Compounds/pharmacology , Ovarian Neoplasms/metabolism , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p53/drug effects
4.
Cancer Lett ; 141(1-2): 85-91, 1999 Jul 01.
Article in English | MEDLINE | ID: mdl-10454247

ABSTRACT

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.


Subject(s)
3-O-Methylglucose/pharmacokinetics , Colonic Neoplasms/metabolism , Deoxyglucose/pharmacokinetics , Hydroxyurea/pharmacology , Cell Division/drug effects , Colonic Neoplasms/pathology , DNA/metabolism , Flow Cytometry , Humans , Time Factors , Tumor Cells, Cultured
5.
Breast Cancer Res Treat ; 53(1): 51-9, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10206072

ABSTRACT

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.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor/metabolism , Breast Neoplasms/drug therapy , Adult , Aged , Biopsy, Needle , Breast Neoplasms/metabolism , Chemotherapy, Adjuvant , Female , Flow Cytometry , Humans , Immunohistochemistry , Ki-67 Antigen/metabolism , Methotrexate/administration & dosage , Middle Aged , Mitoxantrone/administration & dosage , Pilot Projects , Ploidies , Preoperative Care , Prospective Studies , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Tamoxifen/administration & dosage , Treatment Outcome , Tumor Suppressor Protein p53/metabolism
6.
Cytometry ; 33(1): 56-66, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9725559

ABSTRACT

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.


Subject(s)
Growth Inhibitors/pharmacology , Pyridines/pharmacology , Thymidylate Synthase/antagonists & inhibitors , Animals , Bisbenzimidazole , Bromodeoxyuridine , Cell Cycle , Cell Division/drug effects , DNA, Neoplasm/analysis , Flow Cytometry , Folic Acid/analogs & derivatives , Folic Acid Antagonists , Humans , Mice , Propidium , Quinazolines , Staining and Labeling , Tumor Cells, Cultured
7.
Cytometry ; 32(4): 291-300, 1998 Aug 01.
Article in English | MEDLINE | ID: mdl-9701398

ABSTRACT

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.


Subject(s)
Apoptosis , Breast Neoplasms/pathology , Carcinoma/pathology , Adult , Aged , Apoptosis/genetics , Biopsy, Needle , Breast Neoplasms/genetics , Carcinoma/genetics , Cell Separation/methods , DNA Fragmentation , DNA, Neoplasm/analysis , Flow Cytometry/methods , Humans , Middle Aged , S Phase
8.
Int J Cancer ; 77(6): 913-8, 1998 Sep 11.
Article in English | MEDLINE | ID: mdl-9714063

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma/metabolism , Cisplatin/pharmacology , Gene Expression Regulation, Neoplastic , Organoplatinum Compounds/pharmacology , Ovarian Neoplasms/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Tumor Suppressor Protein p53/metabolism , Blotting, Northern , Carcinoma/drug therapy , Carcinoma/genetics , Carcinoma/radiotherapy , DNA Primers , Female , Flow Cytometry , Humans , Immunoblotting , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/radiotherapy , Polymerase Chain Reaction , Tumor Cells, Cultured/metabolism
9.
Nucl Med Biol ; 25(5): 481-5, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9720666

ABSTRACT

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.


Subject(s)
Antimetabolites/metabolism , Antimetabolites/pharmacology , Breast Neoplasms/metabolism , Colonic Neoplasms/metabolism , Deoxyglucose/metabolism , Deoxyglucose/pharmacology , Cell Count , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line , Humans , Tumor Cells, Cultured
10.
Breast Cancer Res Treat ; 48(1): 11-20, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9541185

ABSTRACT

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.


Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Biomarkers, Tumor/analysis , Breast Neoplasms/drug therapy , Carcinoma/drug therapy , Ki-67 Antigen/analysis , Receptors, Cell Surface/analysis , Tamoxifen/therapeutic use , Aged , Aged, 80 and over , Breast Neoplasms/metabolism , Carcinoma/metabolism , Female , Flow Cytometry , Humans , Immunohistochemistry
11.
Cytometry ; 28(3): 264-7, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9222112

ABSTRACT

A staining artefact associated with the terminal deoxynucleotidyl assay for apoptotic cells is described. the Artefact is caused by particulate matter in the reconstituted dried milk used in the washing buffer. We recommend filtering the washing buffer before use.


Subject(s)
Apoptosis , Artifacts , DNA Nucleotidylexotransferase/metabolism , Animals , Buffers , Cells, Cultured , Flow Cytometry , Mice , Milk , Staining and Labeling
12.
Breast Cancer Res Treat ; 44(1): 65-74, 1997 May.
Article in English | MEDLINE | ID: mdl-9164679

ABSTRACT

This study was undertaken to evaluate our ability to detect multiple molecular markers of prognosis and response to treatment in fine needle aspirates (FNA) from patients with primary breast carcinomas. 147 patients with operable primary breast carcinomas who had been recruited to a randomized trial of primary medical therapy (PMT) versus adjuvant chemoendocrine therapy were analysed. FNAs were taken prior to therapy and from this multiple slides were produced using cytospin cytocentrifugation and stored at -80 degrees C for subsequent immunocytochemical analysis (ICA). ICA was performed for oestrogen receptor (ER), progesterone receptor (PgR), p53, Ki67, and Bcl-2. Part of the aspirate was snap frozen and used for flow cytometric analysis of ploidy and S-phase fraction (SPF). In a subgroup of 50 patients who had surgery prior to systemic therapy, as well as FNAs, sections were also taken from paraffin-embedded blocks and stained by ICA for ER, PgR and p53 for validation. In these patients ER was additionally measured by enzyme immunoassay (EIA) from frozen tissue taken at surgery. ER, PgR, p53, Bcl-2, and Ki67 were successfully detected by ICA while ploidy and SPF were successfully measured by flow cytometry from FNA material. The percentage positive values obtained were reasonable and as follows: 74% for ER, 70% for PgR, 36% for p53, 80% for Bcl-2,68% of tumours were aneuploid and 32% diploid. Significant relationships between these measurements were observed in accordance with expectations. The concordance for ER, PgR, and p53 from FNA when compared to ICA of matching histological sections was 91.5%, 75.5%, and 75% respectively. For ER the concordance between measurement by ICA of cytological and histological samples and by EIA of frozen tissue was 82.5% and 84% respectively. These results indicate that multiple molecular markers can be adequately tested on cytological preparations from primary breast tumours. These markers can be used to determine prognosis and predict response to PMT.


Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/chemistry , Adult , Aged , Analysis of Variance , Biomarkers, Tumor/metabolism , Biopsy, Needle/methods , Biopsy, Needle/standards , Breast Neoplasms/diagnosis , Centrifugation , Cytodiagnosis/methods , Cytodiagnosis/standards , Female , Flow Cytometry/methods , Flow Cytometry/standards , Humans , Immunoenzyme Techniques/standards , Immunohistochemistry , Ki-67 Antigen/analysis , Middle Aged , Prognosis , Proto-Oncogene Proteins c-bcl-2/analysis , Receptors, Estrogen/analysis , Receptors, Estrogen/metabolism , Receptors, Progesterone/analysis , Receptors, Progesterone/metabolism , Tumor Suppressor Protein p53/analysis
13.
Clin Cancer Res ; 3(4): 593-600, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9815725

ABSTRACT

Our aim was to determine whether biological molecular markers can predict response to neoadjuvant chemoendocrine therapy in patients with early breast cancer. Ninety patients (median age 56 years; range, 28-69 years) with primary operable breast carcinoma were studied. They were treated with four 3-weekly cycles of chemotherapy with mitozantrone, methotrexate (+/- mitomycin C), and tamoxifen prior to surgery. Fine-needle aspiration was used to obtain samples from patients prior to therapy, and the following parameters were assessed: estrogen receptor (ER), progesterone receptor (PgR), p53, Ki67, Bcl-2, and c-erbB-2 measured by immunocytochemistry, and ploidy and S-phase fraction (SPF) by flow cytometry. The tumors of 78% of the subjects responded (complete response, 9%; partial response, 69%) and 22% did not (no change, 20%; progressive disease, 2%). Response rates according to disease stage and patient age were as follows: T1, 74%; T2, 79%; T3/T4, 78%; age 50, 79% (P = not significant). Response rates for other parameters were as follows: ER-positive, 82%, and -negative, 70%; PgR-positive, 86%, and -negative, 71%; p53-positive, 74%, and -negative, 81%; Bcl-2-positive, 85%, and -negative 61%; c-erbB-2-positive, 57%, and -negative, 93%; Ki67 high, 77%, and low, 81%; SPF high, 77%, and low, 77%; aneuploid, 71%; and diploid, 85%. Only the difference for c-erbB-2 was statistically significant (P = 0.007). A trend for higher response rates to neoadjuvant chemoendocrine therapy for tumors that were positive for ER, PgR, and Bcl-2 was observed but did not reach statistical significance. Tumors negative for c-erbB-2 had a higher response rate, which was statistically significant. In contrast, Ki67, ploidy, SPF, and p53 failed to predict for response.


Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/surgery , Tamoxifen/therapeutic use , Adult , Age Factors , Aged , Antineoplastic Agents, Hormonal/administration & dosage , Biopsy, Needle , Breast Neoplasms/pathology , Breast Neoplasms/radiotherapy , Chemotherapy, Adjuvant , Combined Modality Therapy , Disease Progression , Female , Humans , Menopause , Middle Aged , Mitomycin/administration & dosage , Mitoxantrone/administration & dosage , Neoplasm Staging , Ploidies , Predictive Value of Tests , Proto-Oncogene Proteins c-bcl-2/analysis , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis , S Phase , Tamoxifen/administration & dosage
14.
Br J Cancer ; 74(12): 1935-43, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8980393

ABSTRACT

We have used flow cytometry to study the mechanism of cytotoxic action of a series of ammine/amine Pt(IV) dicarboxylates [ammine diacetatodichloro(cyclohexylamine) platinum(IV), JM216; ammine dibutyratodichloro(cyclohexylamine)platinum(IV), JM221; ammine diacetatodichloro(propylamine)platinum(IV), JM223; ammine dibenzoatodichloro(propylamine)platinum(IV), JM244]. JM216 has been shown to have clinical potential and has recently entered phase II trials. All the compounds caused a slowdown in S-phase transit followed by a block in G2. Cells died either through apoptosis (largely during S-phase) or by failing to overcome the G2 block (some days after treatment). In G2, the cells either divided or enlarged and died. At equitoxic doses, JM216 showed the most apoptotic cells and had the most platinum bound to the DNA; JM244 showed the fewest apoptotic cells and had the least platinum bound to DNA. We suggest that whether apoptosis was triggered or not was governed by the total amount of Pt bound to the DNA; the type of lesion was more important in determining whether a cell became blocked in G2.


Subject(s)
Antineoplastic Agents/pharmacology , Flow Cytometry/methods , Organoplatinum Compounds/pharmacology , Organoplatinum Compounds/toxicity , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Survival/drug effects , DNA, Neoplasm/chemistry , Leukemia L1210/drug therapy , Leukemia L1210/genetics , Mice , Organoplatinum Compounds/chemistry , Platinum/pharmacokinetics
15.
Br J Cancer ; 74(7): 1037-45, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8855971

ABSTRACT

It has been previously demonstrated that cisplatin induces apoptosis in the CH1 human ovarian carcinoma cell line. This study demonstrates that two novel platinum (Pt) analogues JM149 and JM335, which are the cis and trans geometry respectively of ammine(cyclohexylamine)dihydroxodichloroPt(IV), initiate apoptosis in this cell line at physiologically relevant concentrations (IC50 values 2 h drug exposure were 35.3 microM for JM149 and 18.7 microM for JM335). While at equimolar drug concentrations there was a 2-fold higher level of total platinum-DNA adducts following exposure to JM335 vs JM149, at equitoxic concentrations, levels were similar (80 vs 70 pmol Pt mg-1 DNA respectively). Following a 2 h incubation with 2 x IC50 of both drugs, cells rounded up and detached in a time-dependent manner but with the kinetics of apoptosis being more rapid for JM335. The majority of detached cells exhibited morphology associated with apoptosis which was further supported by the presence of a 50 kb fragment detected in DNA lysates prepared from these cells. JM149 induced apoptosis across a range of concentrations (2 x, 5 x and 10 x IC50) with a 50 kb DNA fragment being detected at all concentrations. However, in marked contrast to this, JM335 failed to cause apoptosis at 10 x IC50, the detached cells neither displaying apoptotic morphology nor a detectable 50 kb DNA fragment. Moreover, these detached cells showed evidence of extensive vesiculation while the DNA remained normal in appearance and thus appeared to have died by a non-apoptotic mode. Apoptosis also appeared to be induced to a lesser extent at 5 x IC50 of JM335 as demonstrated by a less intense 50 kb fragment compared with that seen at 2 x IC50. The main cell cycle effect of these drugs (at 2 x IC50) was a slowdown in S-phase traverse during which most but not all of the apoptosis appeared to occur. However, at 5 x IC50 of JM335 cells appeared frozen in all phases of the cell cycle with little progress from G1 to S accompanied by a build-up of cells in G2 indicative of a G2/M block. This difference in cell cycle effect may account for the reduced level of apoptosis at this concentration and a failure to engage apoptosis at higher concentrations. These data suggest that the nature of the platinum drug (and consequently, the nature of resultant DNA damage) may have important implications in determining the rate and mechanism of cell death in this cell line. The cell death effects observed with the trans complex JM335 may correlate with the induction of DNA single-strand breaks in this cell line.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Death , Organoplatinum Compounds/pharmacology , Apoptosis , Cell Cycle/drug effects , DNA, Neoplasm/drug effects , DNA, Neoplasm/metabolism , Drug Screening Assays, Antitumor , Female , Flow Cytometry , Humans , Isomerism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Platinum/metabolism , Time Factors , Tumor Cells, Cultured
16.
Anticancer Res ; 16(3B): 1389-92, 1996.
Article in English | MEDLINE | ID: mdl-8694506

ABSTRACT

Rat sarcoma cells were grown in vitro in tissue culture medium (DMEM) supplemented with 10% foetal calf serum for 5 to 8 days followed by serum withdrawal to produce populations of cells with a variety of cell cycle distributions. Phosphocholine (PCho) and choline content and S + G2 fraction were determined. The phosphocholine content of faster growing populations of serum supplemented cells was higher than the slower growing populations. Choline content was not consistently associated with S + G2 fraction. Serum deprivation was accompanied by a decrease in S + G2 fraction after 24 hours but even after 48 hours PCho content was only slightly decreased.


Subject(s)
Blood Physiological Phenomena , Choline/analysis , Phosphorylcholine/analysis , Sarcoma, Experimental/chemistry , Animals , Culture Media , G2 Phase , Rats , S Phase , Sarcoma, Experimental/pathology
17.
Cytometry ; 21(3): 294-9, 1995 Nov 01.
Article in English | MEDLINE | ID: mdl-8582252

ABSTRACT

The quality of a DNA histogram as recorded in a flow cytometer can often be improved by gating on forward and orthogonal light scatter. This may be helpful when measuring DNA histograms from formalin-fixed, paraffin-embedded material. We report that nuclei from many human tumours scatter more light orthogonally than those from normal stroma. Gating on light scatter enabled the DNA histogram from the tumour to be recorded with reduced contamination from normal cells. In studies of human mammary carcinomas, we have found that this method has the potential to improve the estimation of S phase and the measurement of DNA index (DI) when the DI is close to 1 (near diploid).


Subject(s)
DNA, Neoplasm/analysis , Flow Cytometry/methods , Breast Neoplasms/pathology , Female , Flow Cytometry/instrumentation , Humans , Light , Paraffin Embedding , Scattering, Radiation
18.
Br J Cancer ; 70(6): 1211-6, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7981079

ABSTRACT

Sequential fine-needle aspirates (FNAs) for cytodiagnosis and flow cytometry were taken from 21 patients with primary breast carcinoma at intervals ranging from 1 to 3 months after the commencement of first-line tamoxifen therapy. Nine patients achieved a sustained complete or near complete response over a 3-9 month period. The tumour cells from seven out of nine of these patients were initially aneuploid, while the remaining two patients had diploid tumours. An analysis of sequential FNAs showed that, in three out of the seven aneuploid tumours, only benign epithelial cells could be detected by cytology in the post-tamoxifen sample. In the remaining six cases, including the two diploid tumours, there was no change in ploidy but a reduction in S-phase fraction (SPF) to approximately 50% of the pretreatment level. In all cases, these changes in ploidy or SPF were seen with a mean lead time of 4 months before the tumour had reached clinical complete remission. None of these patients have relapsed after a mean follow-up period of 18 months. The tumours of 12 patients achieved no more than a temporary partial response to primary tamoxifen therapy. In seven out of eight of these cases, which were all initially aneuploid, sequential FNAs during tamoxifen therapy revealed either an increase or no change in the SPF with the tumour remaining aneuploid. In the remaining four cases the tumours were all recorded as being diploid in the pretreatment sample. However, although three of these cases had a temporary partial response to tamoxifen, an aneuploid component was picked up in repeat sequential FNAs with a mean lead time of 5 months before clinical confirmation of eventual disease progression. We conclude that changes in ploidy and SPF detected by flow cytometry may predict initial response and the likelihood of relapse of breast tumours to tamoxifen before clinical changes become evident. These data justify a larger study.


Subject(s)
Breast Neoplasms/diagnosis , Cell Division , Biomarkers , Biopsy, Needle/methods , Breast Neoplasms/drug therapy , DNA, Neoplasm/analysis , Humans , Ploidies , S Phase , Tamoxifen/therapeutic use , Time Factors
19.
NMR Biomed ; 6(5): 318-23, 1993.
Article in English | MEDLINE | ID: mdl-8268064

ABSTRACT

The content of the phospholipid metabolites, phosphocholine, phosphoethanolamine, glycerophosphorylcholine and glycerophosphorylethanolamine was measured in chemical extracts from 46 human breast carcinoma using 31P NMR spectroscopy. Some patients had received therapy prior to tumour resection. The data were therefore stratified into two groups: (i) all tumours; and (ii) untreated tumours. Three indices of tumour proliferation i.e., mitotic index, Ki67 and S-phase fraction were determined on tissue from the same tumours and were found not to correlate with the content of any of these metabolites. In addition oestrogen-receptor status and density, tumour grade and DNA ploidy were obtained on some tumours. The phosphocholine content was higher in high grade tumours when compared with low grade tumours. There was no apparent relationship between DNA ploidy and the content of any of these metabolites. Glycerophosphorylcholine content of oestrogen-receptor positive tumours correlated with receptor density. However, there was no significant difference between receptor positive and negative tumours in the content of any of the phospholipid metabolites measured.


Subject(s)
Breast Neoplasms/chemistry , Carcinoma, Ductal, Breast/chemistry , Phospholipids/analysis , Aneuploidy , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Carcinoma, Ductal, Breast/mortality , Carcinoma, Ductal, Breast/pathology , Carcinoma, Ductal, Breast/therapy , DNA, Neoplasm/analysis , Ethanolamines/analysis , Female , Glycerylphosphorylcholine/analysis , Humans , Ki-67 Antigen , Magnetic Resonance Spectroscopy , Mitotic Index , Neoplasm Proteins/analysis , Nuclear Proteins/analysis , Phosphatidylethanolamines/analysis , Phosphorylcholine/analysis , Prognosis , Receptors, Estrogen/analysis , S Phase , Tissue Extracts
20.
Exp Hematol ; 21(6): 826-8, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8500581

ABSTRACT

We have developed an in vivo model of human chronic myeloid leukemia (CML). A peripheral blood (PB) sample of Philadelphia (Ph) chromosome-positive CML cells in lymphoid blast crisis was transplanted intravenously (IV) into sublethally irradiated severe combined immunodeficient (SCID) mice, and this resulted in engraftment with systemic proliferation. Growth of leukemia was monitored by PB cell morphology and by flow cytometric analysis of murine PB cells labelled with an anti-human leukocyte antigen (HLA) monoclonal antibody. Human cells were first detected in the PB at 4 weeks and comprised a mean of 57% of the total nucleated cells in the PB of these mice by 15 weeks. The Ph chromosome was retained and the population has been successfully passaged. BCR/ABL fusion gene expression was detected in a subsequent passage. Experiments are underway to use this in vivo model to assess the antileukemic activity of BCR/ABL antisense oligonucleotides.


Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Oligonucleotides, Antisense/therapeutic use , Protein-Tyrosine Kinases , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , B-Lymphocytes/pathology , Cloning, Molecular , Disease Models, Animal , Female , Flow Cytometry , Fluorescence , Gene Expression Regulation, Neoplastic/genetics , HLA Antigens/immunology , Homozygote , Humans , Karyotyping , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mice, SCID , Oligonucleotides, Antisense/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-bcr , T-Lymphocytes/pathology , Tumor Cells, Cultured
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