B. Baldetorp - Academia.edu (original) (raw)

Papers by B. Baldetorp

Molecular Cancer Therapeutics

The prognostic and treatment-predictive markers currently in use for breast cancer are commonly b... more The prognostic and treatment-predictive markers currently in use for breast cancer are commonly based on the protein levels of individual genes (e.g., steroid receptors) or aspects of the tumor phenotype, such as histological grade and percentage of cells in the DNA synthesis phase of the cell cycle. Microarrays have previously been used to classify binary classes in breast cancer such as estrogen receptor (ER)-α status. To test whether the properties and specific values of conventional prognostic markers are encoded within tumor gene expression profiles, we have analyzed 48 well-characterized primary tumors from lymph node-negative breast cancer patients using 6728-element cDNA microarrays. In the present study, we used artificial neural networks trained with tumor gene expression data to predict the ER protein values on a continuous scale. Furthermore, we determined a gene expression profile-directed threshold for ER protein level to redefine the cutoff between ER-positive and ER-...

Blood, 1997

The retinoblastoma tumor-suppressor gene, RB, has been implicated in tumor suppression, in regula... more The retinoblastoma tumor-suppressor gene, RB, has been implicated in tumor suppression, in regulation of the cell cycle, and in mediating cell differentiation. RB is necessary for hematopoiesis in mice, and aberrant RB-expression is associated with the progress and prognosis of leukemia. We have used antisense oligonucleotides, established clones stably expressing an antisense RB construct, and also established clones over expressing the retinoblastoma protein (pRb) to study the role of RB expression in monocytic differentiation induced by all-trans retinoic acid (ATRA) or 1-α-25-dihyroxycholecalciferol (Vit D3) in the monoblastic cell line U-937 and erythroid differentiation induced by transforming growth factor β1 (TGFβ1) and hemin in the erythroleukemic cell line K562. A reduction in pRb production in antisense RB-transfected U-937 clones was shown. Antisense oligonucleotides as well as expression of the antisense RB construct suppressed differentiation responses to ATRA or Vit D...

Blood, 1996

Leukemic U-937 cells, which lack normal p53, were stably transfected with a temperature-sensitive... more Leukemic U-937 cells, which lack normal p53, were stably transfected with a temperature-sensitive mutant of p53 to investigate the consequences for growth and differentiation. On induction of wild-type p53 activity at the permissive temperature, some of these cells underwent maturation as judged by the capacity for oxidative burst and the appearance of monocyte related cell surface molecules. Moreover, wild-type p53-expressing cells were more sensitive than p53-negative control cells to induction of differentiation by 1,25- dihydroxycholecalciferol; a twofold to fourfold increase of the fraction of cells showing signs of terminal maturation was observed when wild-type p53-expressing cells were incubated with 1,25- dihydroxycholecalciferol at concentrations that only slightly affected control cells. Whereas wild-type p53 activity per se induced maturation of certain cells, other underwent cell death judging from the reduced capability to exclude trypan blue and the appearance of frag...

Cytometry, 1989

Flow cytometric DNA measurements yield the amount of DNA for each of a large number of cells. A D... more Flow cytometric DNA measurements yield the amount of DNA for each of a large number of cells. A DNA histogram normally consists of a mixture of one or more constellations of G0/G1‐, S‐, G2/M‐phase cells, together with internal standards, debris, background noise, and one or more populations of clumped cells. We have modelled typical DNA histograms as a mixed distribution with Gaussian densities for the G0/G1 and G2/M phases, an S‐phase density, assumed to be uniform between the G0/G1 and G2/M peaks, observed with a Gaussian error, and with Gaussian densities for standards of chicken and trout red blood cells. The debris is modelled as a truncated exponential distribution, and we also have included a uniform background noise distribution over the whole observation interval. We have explored a new approach for maximum‐likelihood analyses of complex DNA histograms by the application of the EM algorithm. This algorithm was used for four observed DNA histograms of varying complexity. Our...

Cytometry, 1998

S-phase fraction (SPF), estimated in the flow cytometric DNA histogram, is a prognostic factor in... more S-phase fraction (SPF), estimated in the flow cytometric DNA histogram, is a prognostic factor in breast cancer. There are, however, some inherent difficulties in the estimation of SPF, such as the influence of debris, aggregates, and normal cells. Most of the available SPF calculation principles try to consider these difficulties, but so far no consensus has been reached with regard to which principle is to be recommended. The aim of the present study was to investigate the prognostic impact of SPF when estimated with different calculation methods in frozen breast cancer samples from 350 patients. Two nonparametric (Rman, Rmin/both rectangle) and three parametric (ACAS/DNA-base, ModFit, and MultiCycle) calculation methods, with and without correction for debris and aggregates, were used. The mean values for SPF varied from 4.3% (ACAS/DNA-base with correction for debris and aggregates) to 9.4% (MultiCycle without any correction for background). The pairwise correlation between methods varied considerably (R = 0.72-0.98). After categorization of SPF values into low SPF (lower two tertiles) and high SPF (upper tertile), all methods yielded statistically significant Pvalues for recurrence-free survival (median follow-up time 67 months), both univariately (0.0004-< 0.0001) and multivariately (0.048-0.0004), after adjusting for nodal status, tumor size, and estrogen receptor status. SPF with background correction did not yield lower P values than SPF without. Regardless of which method was used, SPF showed similar correlations with lymph node involvement, tumor size, and estrogen receptor content. In conclusion, as the mean value of SPF for different calculation methods varies, each laboratory must be restricted to use only one method. Background correction does not seem to improve the prognostic impact of SPF in DNA histograms. Based on the experiences obtained in the present study, S-phase calculation methods without background correction may therefore be the most suitable for routine evaluation of DNA histograms of fresh frozen breast cancer material (ModFit, MultiCycle, and Rman [the latter only for experienced operators]). The nonparametric Rmin, with an automatic setting of the region used for SPF calculation, may be an alternative, but suffers from the disadvantage of not being commercially available yet.

In vivo (Athens, Greece)

An one-step procedure using a nuclear isolation medium containing propidium iodide has been found... more An one-step procedure using a nuclear isolation medium containing propidium iodide has been found to be a suitable preparation technique for flow cytometric DNA analysis in breast cancer samples. In the case of cervical squamous carcinoma, a pretreatment with HCl seems to be a methodological improvement. One advantage with the HCl modification is that some "false" near-diploid cell populations are abolished. These "false" G0/G1 peaks may represent diploid nuclei with a different stainability for propidium iodide compared to normal diploid nuclei. The HCl treatment has, furthermore, the advantage of increasing the elution of nuclei (mean factor of 4.0), especially non-diploid nuclei from higher differentiated squamous carcinomas.

Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology, 1997

To form a methodologic basis for DNA analysis of ductal carcinoma in situ (DCIS) and invasive car... more To form a methodologic basis for DNA analysis of ductal carcinoma in situ (DCIS) and invasive carcinoma (IC) of the breast, including very small lesions, by comparison of flow cytometric (FCM) and image cytometric (ICM) methods for DNA quantitation. The material consisted of 41 DCIS lesions and 26 ICs. FCM DNA analysis of unfixed, frozen samples were compared to (1) FCM of formalin-fixed, paraffin-embedded tissue; (2) ICM of imprints; and (3) ICM of paraffin-embedded tissue sections. FCM of unfixed tissue showed higher DNA measurement precision and a higher number of DNA nondiploid clones as compared to the other three methods. For the classification of DNA diploid/nondiploid cases, high concordance rates were found between the methods. Discordant cases were predominantly DNA neardiploid by FCM of unfixed tissue but DNA diploid by the other methods. The reproducibility of the DNA index (DI) was best in the interval 1.2 < DI < or = 2.2; it was 74% for FCM of fixed tissue and 79...

Scanning electron microscopy, 1984

Experiments recently done in this laboratory have shown that ionizing irradiation can result in a... more Experiments recently done in this laboratory have shown that ionizing irradiation can result in an increase in the ciliary beat frequency of the trachea in vitro even during ongoing irradiation, implying an immediate stimulation of the physiological activity of the cells. This phenomenon is not fully understood and is by itself contradictory to general radio-biological concepts. Ultrastructural investigations on specimens taken immediately after irradiation (10 Gy) have, so far, not shown any changes. It has therefore seemed useful to extend the investigations by examining the effects of in vivo- irradiation (10 Gy) on rabbit trachea during the first 10 days after treatment. The following results have been obtained: Phase I: On days 1-3 after irradiation the beat frequency showed a slight increase (10%) within the irradiated part in comparison with a non-irradiated area of the trachea. No pronounced ultrastructural changes were found these days. Phase II: On days 4-7 after irradiati...

In vivo (Athens, Greece)

We have used a human tumor nude mouse model involving heterotransplantation and serial passage of... more We have used a human tumor nude mouse model involving heterotransplantation and serial passage of an estrogen receptor (ER) positive, progesterone receptor (PgR) negative human endometrial adenocarcinoma. The effects of estradiol treatment on tumor growth, ER activation and PgR induction were investigated two and four years after heterotransplantation. In Experiment I, two years after initial heterotransplantation, tumor growth and proliferative rate showed a dose-related decrease, ER was activated by estradiol treatment (measured through an increased amount of ER bound with high affinity to nuclear element(s) (ERhs) and PgR was induced. Two years later (Experiment II), the amount of ER1s (ER measured in cytosolic fraction) as well as of ERhs was lower than at the beginning of Experiment I. ER could again be activated by estradiol treatment and PgR was also induced. However, in this experiment no effect either of tumor growth or of proliferative rate was observed during the estradio...

Genetic alterations, such as p53 mutations, may affect a tumour&#39;s response to chemotherap... more Genetic alterations, such as p53 mutations, may affect a tumour&#39;s response to chemotherapy. We have treated two human breast cancer cell lines that differ in p53 status with epirubicin in order to study if there are differences in cell cycle kinetic response. MCF-7 cells express wild-type p53, while SK-BR-3 cells express only a mutated form of p53. The transition of cells from one cell cycle stage to another was studied by a bromodeoxyuridine (BrdUrd)-flow cytometry (FCM) method. MCF-7 cells showed a block in the G1 phase after treatment with 50 nM epirubicin for 24 hours, in agreement with the actions of p53 at the G1 checkpoint. SK-BR-3 cells, on the other hand, progressed through the G1 checkpoint and were blocked in late S and G2 phases, presumably due to the activation of a later checkpoint. In addition, studies of the mRNA levels of p53 and its effector gene p21 revealed that although both cell lines expressed p53 mRNA, a marked difference in the mRNA levels of p21 was seen. A dramatic increase in the level of p21 mRNA was seen in epirubicin-treated MCF-7 cells, while no such increase was seen in SK-BR-3 cells.

Physiological Measurement, 1996

Journal of Vascular Research, 2011

G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor 1 (GPER1) is express... more G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor 1 (GPER1) is expressed in the vasculature, but the importance of vascular GPER1 remains to be clarified. Here we investigate effects of the GPER1 agonist G-1 on endothelial cell proliferation using mouse microvascular endothelial bEnd.3 cells. The bEnd.3 cells express mRNA for GPER1. The bEnd.3 cells expressed both ERα and ERβ immunoreactivities. Treatment with G-1 reduced DNA synthesis and cell number with IC50 values of about 2 µM. GPER1 siRNA prevented G-1-induced attenuation of DNA synthesis. G-1 accumulated cells in S and G2 phases of the cell cycle, suggesting that G-1 blocks transition between G2 and M. G-1 had no effect on DNA synthesis in COS-7 cells only weakly expressing GPER1 mRNA. 17β-Estradiol had no effect on DNA synthesis in physiological concentrations (nM). The ER blocker ICI182780 reduced DNA synthesis with similar potency as G-1. Treatment with the ERK/MAP kinase inhibitor PD98059 had no...

JNCI Journal of the National Cancer Institute, 1999

Journal of Cellular Physiology, 1985

The activites of ornithine decarboxylase (ODC) and ODC inhibitory protein (ODC‐antizyme) were stu... more The activites of ornithine decarboxylase (ODC) and ODC inhibitory protein (ODC‐antizyme) were studied in Ehrlich ascites tumor cells, separated according to their position in the cell cycle by centrifugal elutriation. Release and/or synthesis of ODC‐antizyme was induced by putrescine treatment. Each mouse received an intraperitoneal injection of 25 μmoles of putrescine at 0, 1, 2, and 3 hr after tumor transplantation. Tumor cells obtained from putrescine‐treated and control mice at 4 hr after transplantation were separated into fractions representing all phases of the cell cycle. The cell cycle distribution of the tumor cells in each fraction was determined by flow cytometry.In control tumor cells the ODC activity exhibited two maxima; inlate‐G1/early‐S and in late‐S/G2. A marked decrease in ODC activity was observed in mid‐S phase. This decrease coincided with maximum ODC‐antizyme activity (revealed by putrescine treatment), suggesting that ODC‐antizyme is involved in the regulatio...

International Journal of Gynecological Cancer, 1992

The tumor growth phenotype was characterized in relation to concentration of circulating estradio... more The tumor growth phenotype was characterized in relation to concentration of circulating estradiol, estradiol receptor (ER) activation and progesterone receptor (PgR) induction. Ten tumor pieces from an ER and PgR positive human endometrial adenocarcinoma grown in non-oophorectomized nude mice for one year were randomly selected to grow during a preparation phase of 4 weeks either in oophorectomized nude mice - to adapt tumor growth to the absence of estradiol (group A), or in non-oophorectomized nude mice (group B). For the experimental phase, tumor pieces from each group were again randomly assigned to either of two subgroups (i.e., 4 subgroups in all): with estradiol treatment (subgroups A+ and B+), or without (subgroups A- and B-) as control subgroups. There were no differences in take rate or tumor growth rate between the control subgroups (A- vs. B-), indicating tumor growth to be estradiol-independent. The tumor was estradiol-sensitive, however, as tumor growth could be stimulated by estradiol. Despite its estradiol-independence of growth, the tumor&amp;amp;#39;s estradiol-binding capacity varied according to whether the host animals were oophorectomized or not; and despite the similar growth patterns during the experimental phase, the values of high affinty bound ER (ER activation) were greater for tumors grown in non-oophorectomized mice during the preparation phase than for those grown in oophorectomized mice. Thus, our findings show that an ovarain (estradiol) independent but responsive phenotype of tumor growth is present in human endometrial adenocarcinomas growing in nude mice. This growth phenotype may represent an intermediate state of tumor progression to hormone independence and resistance, which has hitherto been observed only in rodent tumors.

International Journal of Gynecological Cancer, 2004

A management program for FIGO stage I-II nonserous, nonclear-cell adenocarcinomas was evaluated. ... more A management program for FIGO stage I-II nonserous, nonclear-cell adenocarcinomas was evaluated. Histopathology and DNA ploidy were used to estimate postoperatively the risk of progression or death of disease and to tailor treatment. The patient material was a population-based consecutive cohort of all women with endometrial cancer in the Southern Swedish Health Care Region diagnosed between June 1993 and June 1996 (n = 553). Of these, 335 were eligible for the management program. Patients estimated to be at low risk were treated by surgery only, while high-risk patients also received vaginal brachytherapy. A large low-risk group consisting of 84% (n = 283) of the patients with an estimated disease-specific 5-year survival of 96% (95% CI = 93-98%) was identified. The high-risk group (n = 52, 16%) showed a worse outcome with an 80% 5-year disease-specific survival (95% CI = 65-89%). The difference in survival between the groups was highly significant (P &lt; 0.0001). Half of the progressions were distant in the high-risk group. Although there is a clear indication for adjuvant therapy for this group, locoregional radiotherapy could be expected to fail in cases with distant progression. Thus, effective systemic treatments need to be developed. Low-risk patients, constituting the majority (84%) of the patients, can be safely treated by surgery only.

Cell Proliferation, 1996

. Growth kinetic data of human tumours, obtained by flow cytometric analysis of cells labelled wi... more . Growth kinetic data of human tumours, obtained by flow cytometric analysis of cells labelled with bromodeoxyuridine (BrdUrd) might provide prognostic information and allow prediction of response to radio‐ and chemotherapy. However, the theoretical models applied for calculation of growth kinetic data are not fully evaluated. The purpose of this study was to investigate the dependence of the estimation of DNA synthesis time (Ts) on sampling time after BrdUrd labelling, using four different mathematical formulas (Begg et al., White &amp; Meistrich, White et al. and Johansson et al.) which have been developed for the evaluation of flow cytometry‐derived data of BrdUrd‐labelled cells. In addition, we have investigated the influence of the growth kinetic properties of the cell populations using two cultured cell lines (one slow and one fast growing), and two heterotransplanted human tumours. The dependence of the estimation of Ts on sampling time was more or less pronounced, depending on the cell population examined and on the formula used. In the fast growing cell line, the estimates of Ts did not vary significantly with sampling time when using the formulas by White et al., whereas in the slow growing cell line, the estimates of Ts did not show any significant dependence on sampling time when using the formula by Johansson et al. In the tumours, the estimation of Ts depended on sampling time with all formulas used, although to different degrees. In one of the tumours, this was mainly caused by the influence of mouse cells, as we demonstrate. Our results indicate that the proliferative characteristics of a cell population should be taken into consideration when choosing a mathematical formula in order to attain Ts values that are independent of sampling time.

Cell Proliferation, 1994

... is the fraction of BrdUrd-labelled cells in G2 + M phases at time t, with the RM(t) value of ... more ... is the fraction of BrdUrd-labelled cells in G2 + M phases at time t, with the RM(t) value of 1. RM(t), and corrected Ts(Tsc): Equation (4) can be resolved with respect to Ts and this leads to a new relationship between t Tse = 1 f J2( 1 - RM(t),) ...

Cell Proliferation, 1994

. Chinese hamster ovary cells were seeded in the absence or presence of the polyamine synthesis i... more . Chinese hamster ovary cells were seeded in the absence or presence of the polyamine synthesis inhibitor 2‐difluoromethylornithine (DFMO). At 14 days after seeding, the cells were labelled for 15–120 min with the thymidine analogue bromo‐deoxyuridine (BrdUrd) and they were then fixed directly after the labelling period. In addition, cells were labelled for 30 min and they were then allowed to progress in BrdUrd‐free medium during a defined post‐labelling time before fixation. An indirect immunofluorescence technique, using the monoclonal BrdUrd antibody and the intercalating stochiometric DNA stain, propidium iodide, was applied to enable quantification of cellular BrdUrd and DNA contents, respectively, by flow cytometry (FCM). By comparing the mean DNA content of BrdUrd‐labelled cells to the mean DNA contents of G1 and G2 cells, a relative measure of the position of the BrdUrd‐labelled cells was obtained (relative movement). Relative movement data, obtained from control and DFMO‐treated cells fixed directly after BrdUrd labelling, indicated that DFMO‐treated cells entered S phase at a normal rate, while their progression through S phase was impaired. DNA histograms of BrdUrd‐labelled control cells fixed directly after labelling showed that most cells were found in early and late S phase, while DNA histograms of BrdUrd‐labelled DFMO‐treated cells showed that most cells were in early S phase, indicating a delayed progression through S phase. Analysis of relative movement of cells that were allowed to progress in BrdUrd‐free medium after labelling showed that DFMO treatment resulted in a significant lengthening of the DNA synthesis time. Labelling index was significantly higher in DFMO‐treated, growth‐inhibited cells than in early plateau phase control cells indicating an S phase accumulation in the former cells.

Molecular Cancer Therapeutics

The prognostic and treatment-predictive markers currently in use for breast cancer are commonly b... more The prognostic and treatment-predictive markers currently in use for breast cancer are commonly based on the protein levels of individual genes (e.g., steroid receptors) or aspects of the tumor phenotype, such as histological grade and percentage of cells in the DNA synthesis phase of the cell cycle. Microarrays have previously been used to classify binary classes in breast cancer such as estrogen receptor (ER)-α status. To test whether the properties and specific values of conventional prognostic markers are encoded within tumor gene expression profiles, we have analyzed 48 well-characterized primary tumors from lymph node-negative breast cancer patients using 6728-element cDNA microarrays. In the present study, we used artificial neural networks trained with tumor gene expression data to predict the ER protein values on a continuous scale. Furthermore, we determined a gene expression profile-directed threshold for ER protein level to redefine the cutoff between ER-positive and ER-...

Blood, 1997

The retinoblastoma tumor-suppressor gene, RB, has been implicated in tumor suppression, in regula... more The retinoblastoma tumor-suppressor gene, RB, has been implicated in tumor suppression, in regulation of the cell cycle, and in mediating cell differentiation. RB is necessary for hematopoiesis in mice, and aberrant RB-expression is associated with the progress and prognosis of leukemia. We have used antisense oligonucleotides, established clones stably expressing an antisense RB construct, and also established clones over expressing the retinoblastoma protein (pRb) to study the role of RB expression in monocytic differentiation induced by all-trans retinoic acid (ATRA) or 1-α-25-dihyroxycholecalciferol (Vit D3) in the monoblastic cell line U-937 and erythroid differentiation induced by transforming growth factor β1 (TGFβ1) and hemin in the erythroleukemic cell line K562. A reduction in pRb production in antisense RB-transfected U-937 clones was shown. Antisense oligonucleotides as well as expression of the antisense RB construct suppressed differentiation responses to ATRA or Vit D...

Blood, 1996

Leukemic U-937 cells, which lack normal p53, were stably transfected with a temperature-sensitive... more Leukemic U-937 cells, which lack normal p53, were stably transfected with a temperature-sensitive mutant of p53 to investigate the consequences for growth and differentiation. On induction of wild-type p53 activity at the permissive temperature, some of these cells underwent maturation as judged by the capacity for oxidative burst and the appearance of monocyte related cell surface molecules. Moreover, wild-type p53-expressing cells were more sensitive than p53-negative control cells to induction of differentiation by 1,25- dihydroxycholecalciferol; a twofold to fourfold increase of the fraction of cells showing signs of terminal maturation was observed when wild-type p53-expressing cells were incubated with 1,25- dihydroxycholecalciferol at concentrations that only slightly affected control cells. Whereas wild-type p53 activity per se induced maturation of certain cells, other underwent cell death judging from the reduced capability to exclude trypan blue and the appearance of frag...

Cytometry, 1989

Flow cytometric DNA measurements yield the amount of DNA for each of a large number of cells. A D... more Flow cytometric DNA measurements yield the amount of DNA for each of a large number of cells. A DNA histogram normally consists of a mixture of one or more constellations of G0/G1‐, S‐, G2/M‐phase cells, together with internal standards, debris, background noise, and one or more populations of clumped cells. We have modelled typical DNA histograms as a mixed distribution with Gaussian densities for the G0/G1 and G2/M phases, an S‐phase density, assumed to be uniform between the G0/G1 and G2/M peaks, observed with a Gaussian error, and with Gaussian densities for standards of chicken and trout red blood cells. The debris is modelled as a truncated exponential distribution, and we also have included a uniform background noise distribution over the whole observation interval. We have explored a new approach for maximum‐likelihood analyses of complex DNA histograms by the application of the EM algorithm. This algorithm was used for four observed DNA histograms of varying complexity. Our...

Cytometry, 1998

S-phase fraction (SPF), estimated in the flow cytometric DNA histogram, is a prognostic factor in... more S-phase fraction (SPF), estimated in the flow cytometric DNA histogram, is a prognostic factor in breast cancer. There are, however, some inherent difficulties in the estimation of SPF, such as the influence of debris, aggregates, and normal cells. Most of the available SPF calculation principles try to consider these difficulties, but so far no consensus has been reached with regard to which principle is to be recommended. The aim of the present study was to investigate the prognostic impact of SPF when estimated with different calculation methods in frozen breast cancer samples from 350 patients. Two nonparametric (Rman, Rmin/both rectangle) and three parametric (ACAS/DNA-base, ModFit, and MultiCycle) calculation methods, with and without correction for debris and aggregates, were used. The mean values for SPF varied from 4.3% (ACAS/DNA-base with correction for debris and aggregates) to 9.4% (MultiCycle without any correction for background). The pairwise correlation between methods varied considerably (R = 0.72-0.98). After categorization of SPF values into low SPF (lower two tertiles) and high SPF (upper tertile), all methods yielded statistically significant Pvalues for recurrence-free survival (median follow-up time 67 months), both univariately (0.0004-&lt; 0.0001) and multivariately (0.048-0.0004), after adjusting for nodal status, tumor size, and estrogen receptor status. SPF with background correction did not yield lower P values than SPF without. Regardless of which method was used, SPF showed similar correlations with lymph node involvement, tumor size, and estrogen receptor content. In conclusion, as the mean value of SPF for different calculation methods varies, each laboratory must be restricted to use only one method. Background correction does not seem to improve the prognostic impact of SPF in DNA histograms. Based on the experiences obtained in the present study, S-phase calculation methods without background correction may therefore be the most suitable for routine evaluation of DNA histograms of fresh frozen breast cancer material (ModFit, MultiCycle, and Rman [the latter only for experienced operators]). The nonparametric Rmin, with an automatic setting of the region used for SPF calculation, may be an alternative, but suffers from the disadvantage of not being commercially available yet.

In vivo (Athens, Greece)

An one-step procedure using a nuclear isolation medium containing propidium iodide has been found... more An one-step procedure using a nuclear isolation medium containing propidium iodide has been found to be a suitable preparation technique for flow cytometric DNA analysis in breast cancer samples. In the case of cervical squamous carcinoma, a pretreatment with HCl seems to be a methodological improvement. One advantage with the HCl modification is that some "false" near-diploid cell populations are abolished. These "false" G0/G1 peaks may represent diploid nuclei with a different stainability for propidium iodide compared to normal diploid nuclei. The HCl treatment has, furthermore, the advantage of increasing the elution of nuclei (mean factor of 4.0), especially non-diploid nuclei from higher differentiated squamous carcinomas.

Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology, 1997

To form a methodologic basis for DNA analysis of ductal carcinoma in situ (DCIS) and invasive car... more To form a methodologic basis for DNA analysis of ductal carcinoma in situ (DCIS) and invasive carcinoma (IC) of the breast, including very small lesions, by comparison of flow cytometric (FCM) and image cytometric (ICM) methods for DNA quantitation. The material consisted of 41 DCIS lesions and 26 ICs. FCM DNA analysis of unfixed, frozen samples were compared to (1) FCM of formalin-fixed, paraffin-embedded tissue; (2) ICM of imprints; and (3) ICM of paraffin-embedded tissue sections. FCM of unfixed tissue showed higher DNA measurement precision and a higher number of DNA nondiploid clones as compared to the other three methods. For the classification of DNA diploid/nondiploid cases, high concordance rates were found between the methods. Discordant cases were predominantly DNA neardiploid by FCM of unfixed tissue but DNA diploid by the other methods. The reproducibility of the DNA index (DI) was best in the interval 1.2 < DI < or = 2.2; it was 74% for FCM of fixed tissue and 79...

Scanning electron microscopy, 1984

Experiments recently done in this laboratory have shown that ionizing irradiation can result in a... more Experiments recently done in this laboratory have shown that ionizing irradiation can result in an increase in the ciliary beat frequency of the trachea in vitro even during ongoing irradiation, implying an immediate stimulation of the physiological activity of the cells. This phenomenon is not fully understood and is by itself contradictory to general radio-biological concepts. Ultrastructural investigations on specimens taken immediately after irradiation (10 Gy) have, so far, not shown any changes. It has therefore seemed useful to extend the investigations by examining the effects of in vivo- irradiation (10 Gy) on rabbit trachea during the first 10 days after treatment. The following results have been obtained: Phase I: On days 1-3 after irradiation the beat frequency showed a slight increase (10%) within the irradiated part in comparison with a non-irradiated area of the trachea. No pronounced ultrastructural changes were found these days. Phase II: On days 4-7 after irradiati...

In vivo (Athens, Greece)

We have used a human tumor nude mouse model involving heterotransplantation and serial passage of... more We have used a human tumor nude mouse model involving heterotransplantation and serial passage of an estrogen receptor (ER) positive, progesterone receptor (PgR) negative human endometrial adenocarcinoma. The effects of estradiol treatment on tumor growth, ER activation and PgR induction were investigated two and four years after heterotransplantation. In Experiment I, two years after initial heterotransplantation, tumor growth and proliferative rate showed a dose-related decrease, ER was activated by estradiol treatment (measured through an increased amount of ER bound with high affinity to nuclear element(s) (ERhs) and PgR was induced. Two years later (Experiment II), the amount of ER1s (ER measured in cytosolic fraction) as well as of ERhs was lower than at the beginning of Experiment I. ER could again be activated by estradiol treatment and PgR was also induced. However, in this experiment no effect either of tumor growth or of proliferative rate was observed during the estradio...

Genetic alterations, such as p53 mutations, may affect a tumour&#39;s response to chemotherap... more Genetic alterations, such as p53 mutations, may affect a tumour&#39;s response to chemotherapy. We have treated two human breast cancer cell lines that differ in p53 status with epirubicin in order to study if there are differences in cell cycle kinetic response. MCF-7 cells express wild-type p53, while SK-BR-3 cells express only a mutated form of p53. The transition of cells from one cell cycle stage to another was studied by a bromodeoxyuridine (BrdUrd)-flow cytometry (FCM) method. MCF-7 cells showed a block in the G1 phase after treatment with 50 nM epirubicin for 24 hours, in agreement with the actions of p53 at the G1 checkpoint. SK-BR-3 cells, on the other hand, progressed through the G1 checkpoint and were blocked in late S and G2 phases, presumably due to the activation of a later checkpoint. In addition, studies of the mRNA levels of p53 and its effector gene p21 revealed that although both cell lines expressed p53 mRNA, a marked difference in the mRNA levels of p21 was seen. A dramatic increase in the level of p21 mRNA was seen in epirubicin-treated MCF-7 cells, while no such increase was seen in SK-BR-3 cells.

Physiological Measurement, 1996

Journal of Vascular Research, 2011

G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor 1 (GPER1) is express... more G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor 1 (GPER1) is expressed in the vasculature, but the importance of vascular GPER1 remains to be clarified. Here we investigate effects of the GPER1 agonist G-1 on endothelial cell proliferation using mouse microvascular endothelial bEnd.3 cells. The bEnd.3 cells express mRNA for GPER1. The bEnd.3 cells expressed both ERα and ERβ immunoreactivities. Treatment with G-1 reduced DNA synthesis and cell number with IC50 values of about 2 µM. GPER1 siRNA prevented G-1-induced attenuation of DNA synthesis. G-1 accumulated cells in S and G2 phases of the cell cycle, suggesting that G-1 blocks transition between G2 and M. G-1 had no effect on DNA synthesis in COS-7 cells only weakly expressing GPER1 mRNA. 17β-Estradiol had no effect on DNA synthesis in physiological concentrations (nM). The ER blocker ICI182780 reduced DNA synthesis with similar potency as G-1. Treatment with the ERK/MAP kinase inhibitor PD98059 had no...

JNCI Journal of the National Cancer Institute, 1999

Journal of Cellular Physiology, 1985

The activites of ornithine decarboxylase (ODC) and ODC inhibitory protein (ODC‐antizyme) were stu... more The activites of ornithine decarboxylase (ODC) and ODC inhibitory protein (ODC‐antizyme) were studied in Ehrlich ascites tumor cells, separated according to their position in the cell cycle by centrifugal elutriation. Release and/or synthesis of ODC‐antizyme was induced by putrescine treatment. Each mouse received an intraperitoneal injection of 25 μmoles of putrescine at 0, 1, 2, and 3 hr after tumor transplantation. Tumor cells obtained from putrescine‐treated and control mice at 4 hr after transplantation were separated into fractions representing all phases of the cell cycle. The cell cycle distribution of the tumor cells in each fraction was determined by flow cytometry.In control tumor cells the ODC activity exhibited two maxima; inlate‐G1/early‐S and in late‐S/G2. A marked decrease in ODC activity was observed in mid‐S phase. This decrease coincided with maximum ODC‐antizyme activity (revealed by putrescine treatment), suggesting that ODC‐antizyme is involved in the regulatio...

International Journal of Gynecological Cancer, 1992

The tumor growth phenotype was characterized in relation to concentration of circulating estradio... more The tumor growth phenotype was characterized in relation to concentration of circulating estradiol, estradiol receptor (ER) activation and progesterone receptor (PgR) induction. Ten tumor pieces from an ER and PgR positive human endometrial adenocarcinoma grown in non-oophorectomized nude mice for one year were randomly selected to grow during a preparation phase of 4 weeks either in oophorectomized nude mice - to adapt tumor growth to the absence of estradiol (group A), or in non-oophorectomized nude mice (group B). For the experimental phase, tumor pieces from each group were again randomly assigned to either of two subgroups (i.e., 4 subgroups in all): with estradiol treatment (subgroups A+ and B+), or without (subgroups A- and B-) as control subgroups. There were no differences in take rate or tumor growth rate between the control subgroups (A- vs. B-), indicating tumor growth to be estradiol-independent. The tumor was estradiol-sensitive, however, as tumor growth could be stimulated by estradiol. Despite its estradiol-independence of growth, the tumor&amp;amp;#39;s estradiol-binding capacity varied according to whether the host animals were oophorectomized or not; and despite the similar growth patterns during the experimental phase, the values of high affinty bound ER (ER activation) were greater for tumors grown in non-oophorectomized mice during the preparation phase than for those grown in oophorectomized mice. Thus, our findings show that an ovarain (estradiol) independent but responsive phenotype of tumor growth is present in human endometrial adenocarcinomas growing in nude mice. This growth phenotype may represent an intermediate state of tumor progression to hormone independence and resistance, which has hitherto been observed only in rodent tumors.

International Journal of Gynecological Cancer, 2004

A management program for FIGO stage I-II nonserous, nonclear-cell adenocarcinomas was evaluated. ... more A management program for FIGO stage I-II nonserous, nonclear-cell adenocarcinomas was evaluated. Histopathology and DNA ploidy were used to estimate postoperatively the risk of progression or death of disease and to tailor treatment. The patient material was a population-based consecutive cohort of all women with endometrial cancer in the Southern Swedish Health Care Region diagnosed between June 1993 and June 1996 (n = 553). Of these, 335 were eligible for the management program. Patients estimated to be at low risk were treated by surgery only, while high-risk patients also received vaginal brachytherapy. A large low-risk group consisting of 84% (n = 283) of the patients with an estimated disease-specific 5-year survival of 96% (95% CI = 93-98%) was identified. The high-risk group (n = 52, 16%) showed a worse outcome with an 80% 5-year disease-specific survival (95% CI = 65-89%). The difference in survival between the groups was highly significant (P &lt; 0.0001). Half of the progressions were distant in the high-risk group. Although there is a clear indication for adjuvant therapy for this group, locoregional radiotherapy could be expected to fail in cases with distant progression. Thus, effective systemic treatments need to be developed. Low-risk patients, constituting the majority (84%) of the patients, can be safely treated by surgery only.

Cell Proliferation, 1996

. Growth kinetic data of human tumours, obtained by flow cytometric analysis of cells labelled wi... more . Growth kinetic data of human tumours, obtained by flow cytometric analysis of cells labelled with bromodeoxyuridine (BrdUrd) might provide prognostic information and allow prediction of response to radio‐ and chemotherapy. However, the theoretical models applied for calculation of growth kinetic data are not fully evaluated. The purpose of this study was to investigate the dependence of the estimation of DNA synthesis time (Ts) on sampling time after BrdUrd labelling, using four different mathematical formulas (Begg et al., White &amp; Meistrich, White et al. and Johansson et al.) which have been developed for the evaluation of flow cytometry‐derived data of BrdUrd‐labelled cells. In addition, we have investigated the influence of the growth kinetic properties of the cell populations using two cultured cell lines (one slow and one fast growing), and two heterotransplanted human tumours. The dependence of the estimation of Ts on sampling time was more or less pronounced, depending on the cell population examined and on the formula used. In the fast growing cell line, the estimates of Ts did not vary significantly with sampling time when using the formulas by White et al., whereas in the slow growing cell line, the estimates of Ts did not show any significant dependence on sampling time when using the formula by Johansson et al. In the tumours, the estimation of Ts depended on sampling time with all formulas used, although to different degrees. In one of the tumours, this was mainly caused by the influence of mouse cells, as we demonstrate. Our results indicate that the proliferative characteristics of a cell population should be taken into consideration when choosing a mathematical formula in order to attain Ts values that are independent of sampling time.

Cell Proliferation, 1994

... is the fraction of BrdUrd-labelled cells in G2 + M phases at time t, with the RM(t) value of ... more ... is the fraction of BrdUrd-labelled cells in G2 + M phases at time t, with the RM(t) value of 1. RM(t), and corrected Ts(Tsc): Equation (4) can be resolved with respect to Ts and this leads to a new relationship between t Tse = 1 f J2( 1 - RM(t),) ...

Cell Proliferation, 1994

. Chinese hamster ovary cells were seeded in the absence or presence of the polyamine synthesis i... more . Chinese hamster ovary cells were seeded in the absence or presence of the polyamine synthesis inhibitor 2‐difluoromethylornithine (DFMO). At 14 days after seeding, the cells were labelled for 15–120 min with the thymidine analogue bromo‐deoxyuridine (BrdUrd) and they were then fixed directly after the labelling period. In addition, cells were labelled for 30 min and they were then allowed to progress in BrdUrd‐free medium during a defined post‐labelling time before fixation. An indirect immunofluorescence technique, using the monoclonal BrdUrd antibody and the intercalating stochiometric DNA stain, propidium iodide, was applied to enable quantification of cellular BrdUrd and DNA contents, respectively, by flow cytometry (FCM). By comparing the mean DNA content of BrdUrd‐labelled cells to the mean DNA contents of G1 and G2 cells, a relative measure of the position of the BrdUrd‐labelled cells was obtained (relative movement). Relative movement data, obtained from control and DFMO‐treated cells fixed directly after BrdUrd labelling, indicated that DFMO‐treated cells entered S phase at a normal rate, while their progression through S phase was impaired. DNA histograms of BrdUrd‐labelled control cells fixed directly after labelling showed that most cells were found in early and late S phase, while DNA histograms of BrdUrd‐labelled DFMO‐treated cells showed that most cells were in early S phase, indicating a delayed progression through S phase. Analysis of relative movement of cells that were allowed to progress in BrdUrd‐free medium after labelling showed that DFMO treatment resulted in a significant lengthening of the DNA synthesis time. Labelling index was significantly higher in DFMO‐treated, growth‐inhibited cells than in early plateau phase control cells indicating an S phase accumulation in the former cells.