Fate map of early avian cardiac progenitor cells.

Cardiogenic fate maps are used to address questions on commitment, differentiation, morphogenesis and organogenesis of the heart. Recently, the accuracy of classical cardiogenic fate maps has been questioned, raising concerns about the conclusions drawn in studies based on these maps. We present accurate fate maps of the heart-forming region (HFR) in avian embryos and show that the putative cardiogenic molecular markers Bmp2 and Nkx2.5 do not govern the boundaries of the HFR as suggested in the literature. Moreover, this paper presents the first fate map of the HFR at stage 4 and addresses a void in the literature concerning rostrocaudal patterning of heart cells between stages 4 and 8.

UCN-01 dose-dependent inhibition of normal hyperproliferative cells in mice.

UCN-01 is a hydroxylated derivative of staurosporine and a potent protein kinase C (PKC) inhibitor. Interest in the potential usefulness of this compound as an anticancer drug stems mainly from its unique anti-signaling, growth-arresting properties on tumor cells. This include activation of CDC2 kinase (CDK1) which interacts with either cyclin A or cyclin B1 at the G1 or G2/M border, suggeting that this event is one of the major consequences of the drug action on eukaryotic cells. Nonetheless, the antiproliferative activity of UCN-01 on normal rapidly dividing cells (intestinal epithelial and bone marrow cells) is not well documented. Thus, the main objective of this study was to investigate the in vivo antiproliferative activity of UCN-01 on these normal hyperproliferative cells and evaluate whether cellular response to UCN-01 could be modulated in the presence of DNA damage. Mice were injected i.m. with a single dose of UCN-01 (2.5 mg/kg-20 mg/kg) followed 3 and 24 h later by in vivo BrdU labeling for 1 h. At autopsy, bone marrow cells were collected and fixed for dual parameter BrdU/DNA flow cytometry. Different regions of the gut were also fixed for immunoperoxidase BrdU assays. Newly replicated cells were mainly located in the lower compartments of the crypt columns and were scored for BrdU stained nuclei using an image analysis system. A comparison between groups showed that 5 mg/kg UCN-01 induced inhibition in BrdU incorporation at 3 and 24 h, as compared to the other groups injected with various doses of UCN-01. Flow cytometric analysis of bone marrow cells stained with fluorescein tagged anti-BrdU (FITC) along with propidium iodide (PI) also showed inhibition in BrdU incorporation of S phase fraction cells in mice treated with 5 mg/kg UCN-01. These bone marrow cells were arrested primarily in the G1 phase of the cell cycle. The colony-forming unit (CFU) assay of the bone marrow cells was then used to determine the level of drug interaction of UCN-01 and, topotecan, a topoisomerase I inhibitor, at a fixed dose ratio. An antagonistic drug interaction (CI > 1) was observed as determined by the median-effect analysis. However, an additive interaction (CI = 1) was obtained with the use of camptothecin or 10,11-methylenedioxycamptothecin and UCN-01. The results of the in vitro drug interaction with UCN-01 may predict protection from topotecan-induced bone marrow toxicity.

Abrogation of cell cycle checkpoint controls during malignant transformation of syrian hamster embryo cells is associated with decreased sensitivity to apoptosis.

Malachite green (MG), consisting of green crystals with a metallic luster, is highly soluble in water, cytotoxic to various mammalian cells, and may act as a liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have reported earlier the malignant transformation of Syrian hamster embryo (SHE) cells by MG. In this study, we investigated the effects of MG on flow cytometric cell cycle phase distribution of normal and MG-transformed SHE cells in asynchronous and synchronous cell populations. DNA flow cytometric analysis indicated that culturing cells for 48 hours in a medium containing MG 0.1 microg/mL induced G2/M arrest in normal cells. Malignant-transformed cells showed no such accumulation of cells at the G2/M phase of the cell cycle in response to MG. Synchronization studies indicated that in the control, both in the presence and absence of MG, cells followed a normal cell cycle pattern up to 16 hours. After 16 hours, in the absence of MG, cells continued a normal cell cycle, whereas in the presence of MG they accumulated at the G2/M phase of the cell cycle. This pattern of accumulation of cells at the G2/M checkpoint control was not observed in either untreated or MG-treated transformed cells. We also studied the effects of MG on the induction of apoptosis using flow cytometric FSC/SSC scatter plots in normal and transformed SHE cells. Flow cytometric analysis showed a dose- and time-dependent induction of apoptosis by MG in control cells, whereas induction of apoptosis by MG was marginal in transformed cells. In the present study, we demonstrated the efficient operation of the G2/M checkpoint control, apoptosis in control SHE cells, the abrogation of checkpoint controls, and decreased sensitivity to apoptosis in transformed SHE cells.

Extensibility of isoforms of cardiac titin: variation in contour length of molecular subsegments provides a basis for cellular passive stiffness diversity.

Titin is a giant polypeptide that spans between the Z- and M-lines of the cardiac muscle sarcomere and that develops force when extended. This force arises from titin's extensible I-band region, which consists mainly of three segment types: serially linked immunoglobulin-like domains (Ig segments), interrupted by the PEVK segment, and the N2B unique sequence. Recently it was reported that the myocardium of large mammals co-expresses small (N2B) and large (N2BA) cardiac isoforms and that the passive stiffness of cardiac myocytes varies with the isoform expression ratio. To understand the molecular basis of the differences in passive stiffness we investigated titin's extensibility in bovine atrium, which expresses predominantly N2BA titin, and compared it to that of rat, which expresses predominantly N2B titin. Immunoelectron microscopy was used with antibodies that flank the Ig segments, the PEVK segment, and the unique sequence of the N2B element. The extension of the various segments was then determined as a function of sarcomere length (SL). When slack sarcomeres of bovine atrium were stretched, the PEVK segment extended much more steeply and the unique N2B sequence less steeply than in rat, while the Ig segments behaved similarly in both species. However, the extensions normalized with the segment's contour length (i.e., the fractional extensions) of Ig, PEVK, and unique sequence segments all increase less steeply with SL in cow than in rat. Considering that fractional extension determines the level of entropic force, these differences in fractional extension are expected to result in shallow and steep passive force-SL curves in myocytes that express high levels of N2BA and N2B titin, respectively. Thus, the findings provide a molecular basis for passive stiffness diversity.

Genes in the first and fourth inversions of the mouse t complex synergistically mediate sperm capacitation and interactions with the oocyte.

The t haplotypes (t) are recent evolutionary derivatives of an alternate form of the mouse t complex region located at the proximal end of chromosome 17. This variant form of approximately 1% of the mouse genome is a source of mutations altering numerous sperm functions crucial for fertilization. Males that carry two t haplotypes (t/t) are invariably sterile. t haplotypes contain four inversions relative to the wild-type t complex (+), so that in matings involving a +/t heterozygote, t is usually transmitted as a single unit. However, rare recombinants have been recovered, which carry only part of the t genotype and express only some of the t-dependent phenotypes. Use of these partial t haplotypes in genetic crosses has resulted in the general location of the two major t male sterility factors, S1 and S2, within inversions 1 and 4, respectively. Since sterility can result from a plethora of sperm defects, we have made a detailed study of various functional parameters of sperm from mice carrying S1 or S2 heterozygously or homozygously or in combination. Both S1 and S2 contain mutations altering sperm functions, including motility, capacitation, binding to the zona pellucida, binding to the oocyte membrane, and penetration of the zona pellucida-free oocyte. Therefore it seems clear that each of these factors contains multiple genes contributing to sterility. Furthermore, our results indicate that genes within S1 interact with genes in S2 for all sperm functions examined. However, S1 and S2 genes affecting motility interact in a purely additive fashion, while S1 and S2 genes affecting most other sperm characteristics interact in a synergistic manner. Additionally, the patterns of synergism between S1 and S2 for abnormalities in capacitation, sperm-oolemma binding, and zona-free oocyte penetration are nearly identical. This suggests that these three defects are caused by mutation of the same gene within each sterility factor. These findings will not only be instrumental in matching the various t haplotype sperm defects to candidate genes for S1 and S2, but will facilitate a more comprehensive understanding of the cellular and genetic mechanisms underlying t haplotype male sterility.

UCN-01 and camptothecin induce DNA double-strand breaks in p53 mutant tumor cells, but not in normal or p53 negative epithelial cells.

Previous research has shown synergistic growth inhibition between UCN-01 and camptothecin (CPT) in tumor cells with mutant p53 versus tumor cells with wild-type p53. To determine the possible role of p53 in this drug combination, we tested the hypothesis that the synergistic growth inhibition is due to the absence of p53, and can result from the induction of DNA double-strand breaks (DSBs). Experiments were performed with the use of normal human mammary epithelial cells (HMEC); HMEC transfected with HPV16 E6 protein which inactivates p53 (HE6), or p53-mutant MDA-MB-231 tumor cells. CPT, UCN-01, or a 1:1 combination of both, in either HMEC or HE6 cells did not induce DSBs. In contrast, simultaneous treatment of MDA-MB-231 cells with both UCN-01 and CPT induced significant levels of DSBs while treatment with either drug alone did not. While UCN-01 was surprisingly potent against HMEC, the growth inhibition was only additive between UCN-01 and CPT against these cells. HE6 cells were much less sensitive than HMEC to UCN-01 and slightly less sensitive to the combined treatment with UCN-01 and CPT. The drug combination was synergistic against HE6 cells, due to their lower sensitivity to UCN-01. Unlike what was observed previously in MDA-MB-231 cells, UCN-01 did not abrogate CPT-induced inhibition of DNA synthesis in either HMEC or HE6 cells. These data indicate that synergistic growth inhibition by UCN-01 and CPT against p53 mutant MDA-MB-231 tumor cells may be due to induction of DSBs however the loss of p53 function alone does not sensitize normal cells to the combination of both drugs.

Mechanical properties of titin isoforms.

Titin is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the sarcomere. As a result of differential splicing, length variants of titin are expressed in different skeletal and cardiac muscles. Here we first briefly review some of our previous work that has revealed that titin develops force in sarcomeres either stretched beyond their slack length (passive force) or shortened to below the slack length (restoring force) and that titin's force underlies a large fraction of the diastolic force of cardiac muscle. Next we present our mechanical and immunoelectron microscopical (IEM) studies of skeletal and cardiac muscles that express titin isoforms. The previously deduced molecular properties of titin were used to model titin's extensible region in the sarcomere as serially linked WLCs: rigid segments (containing folded Ig/Fn domains) and more flexible segments (PEVK segment). The model was tested on skeletal muscle fibers that express titin isoforms with tandem Ig and PEVK length variants. The model adequately predicts titin's behavior along a wide sarcomere length range in skeletal muscle, but at long sarcome lengths (SLs), predicted forces are much higher than those determined experimentally. IEM reveals that this may result from Ig domain unfolding. Experiments were also performed on cardiac myocytes from mouse and cow that express predominantly a small cardiac titin isoform (N2B titin) or a large isoform (N2BA titin), respectively. The passive tension-SL relation of myocytes was found to increase more steeply with SL in mouse than in cow. IEM revealed an additional source of extensibility within both of these cardiac titins: the unique N2B sequence (absent in skeletal muscle). Furthermore, the PEVK segment of the N2BA isoform extended to a maximal length of approximately 200 nm, as opposed to approximately 60 nm for the N2B isoform. We propose that, along the physiological SL range, the long PEVK segment found in N2BA titins results in a low PEVK fractional extension and that this underlies the lower passive tensions of N2BA-expressing cow myocytes.

Malachite green induced malignant transformation of Syrian hamster embryo (SHE) cells in primary culture: transformation is associated with enhanced expression of altered p53, bcl-2 and decreased sensitivity to apoptosis.

Malachite green (MG) consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have previously reported the malignant transformation of Syrian hamster embryo (SHE) cells by MG. In this study, we have made an attempt to study the effects of MG on the induction of apoptosis in normal and MG transformed Syrian hamster embryo cells and the expression of altered p53 and bcl-2 immunohistochemically. Induction of apoptosis was detected by flow cytometry on the basis of G0/G1 hypodiploid peak, Tunel assay and DNA ladder pattern. Flow cytometric analysis showed a dose and time dependent induction of apoptosis by MG in control cells whereas induction of apoptosis by MG was marginal in transformed cells. Tunel assay and DNA ladder pattern also showed decreased apoptosis in transformed cells by MG compared to controls. Immunostaining studies showed intense staining for p53 in transformed cells whereas no staining was observed in control cells. Also transformed cells showed overexpression of bcl-2 with exclusive nuclear localization compared to controls which did not show staining. The present study indicated that MG transformed Syrian hamster embryo cells have decreased sensitivity to apoptosis compared to normal cells and overexpression of altered p53 and bcl-2 seems to be conferring resistance to MG induced apoptosis.

An improved mouse sperm-oocyte plasmalemma binding assay: studies on characteristics of sperm binding in medium with or without glucose.

Sperm binding to the oocyte plasmalemma is crucial to subsequent steps in fertilization. However, the usual in vitro assay for sperm-oocyte binding does not distinguish between nonspecific attachment and specific binding leading to fusion and penetration. Since zona pellucida-free pronuclear zygotes should not bind sperm (because of the block to polyspermy at the level of the oocyte membrane), a procedure has been developed to remove virtually all sperm from zona-free pronuclear zygotes (2PNZ). After six washes with a 90-microm-bore pipette, there were 0.5 +/- 0.2 sperm/2PNZ (n = 83). Therefore, these washing conditions were used to define sperm-oocyte binding. The relationship of binding to oocyte penetration was determined for outbred mouse oocytes coincubated in complete medium with (B6 x 129)F1 hybrid sperm (10(7)/ml). Binding was maximal (29 +/- 5 sperm per oocyte) during the first 30 minutes but decreased significantly to 4.6 +/- 1.4 by 60 minutes of coincubation (over 10 trials). Oocyte penetration was 99 +/- 1% by 30 minutes, while the number of decondensed sperm nuclei per oocyte increased significantly to 7.5 +/- 0.6 at 60 minutes. These data suggest that the block to polyspermy involves detachment of bound sperm. Similar coincubations were carried out in medium without glucose (NoG), as this medium has been reported to inhibit fusion without affecting binding. However, binding was only 11 +/- 2 at 30 minutes but increased to 25 +/- 4 at 60 minutes, suggesting that binding was retarded in NoG. When gametes were coincubated in NoG for 5 hours, about half of the oocytes were penetrated, suggesting that the lack of glucose did not inhibit fusion but instead delayed it. These data suggest that if sperm binding is to be determined in complete medium, the time of the block to polyspermy should be determined prior to selecting the appropriate time to assay binding. Furthermore, using the same coincubation period for the binding assay in control and treated sperm may not be appropriate if the treatment alters the time of maximal binding.

Expression of Fas and Fas ligand in Hodgkin's disease.

Fas and Fas ligand expression were investigated in twenty two cases of classical Hodgkin's disease (HD) by immunohistochemistry. While Reed-Sternberg (RS) cells in 7/22 (32%) cases expressed Fas ligand, reactive lymphoid cells expressed Fas ligand in only 2 (9%) cases. In 20/22 (91%) cases, the RS cells expressed Fas. A higher proportion of RS cells in the nodular sclerosis subtype expressed Fas as compared to the mixed cellularity subtype. In 18/22 (82%) cases, Fas expression was also noted in the reactive lymphoid cells. In eight cases, the reactive lymphoid cells were also analyzed by flow cytometry and a majority of them were CD4+CD45RO+. Most of these activated T-cells expressed Fas but were negative for Fas Ligand. To investigate the co-expression of Fas and Fas Ligand in the RS cells, six cases were subjected to Fas and Fas ligand immunostaining on consecutive sections. The co-expression was documented in the RS cells in four of six cases. These six cases with expression of both Fas and Fas ligand were investigated for the incidence of apoptosis. There was no statistically significant relationship between expression of Fas on reactive cells, expression of FasL on RS cells and the proportion of apoptotic reactive cells. In all these cases apoptosis was not observed in the RS cells. Thus Fas - FasL interactions may not lead to apoptosis of the RS cells.

Flow cytometric analysis of estrogen, progesterone receptor expression and DNA content in formalin-fixed, paraffin-embedded human breast tumors.

Flow cytometric analysis of estrogen (ER) and progesterone (PgR) receptor expression in archival human breast tumors is relatively difficult. We have used enzyme digestion and microwave antigen retrieval procedures for multiparametric flow cytometric analysis of ER and PgR expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded primary breast tumors. Deparaffinized rehydrated tissue sections treated with pepsin were subjected to microwave irradiation for unmasking of ER and PgR antigenic sites. Biotinylated ER antibody and streptavidin-fluorescein isothiocyanate (FITC) were used for ER labeling and PgR antibody with phycoerythrin labeled goat anti-mouse antibody was used for PgR labeling. Counter staining with propidium iodide-RNase was used for determination of cellular DNA content. Our results show that enzyme digestion and microwave treatment of formalin-fixed, paraffin-embedded breast tumors can be successfully used for the multiparametric analysis of nuclear hormone receptor expression and DNA content by flow cytometry.

Malignant transformation of Syrian hamster embryo (SHE) cells in culture by malachite green: an agent of environmental importance.

Malachite green (MG), consisting of green crystals with a metallic lustre, is very soluble in water and is highly cytotoxic to mammalian cells in culture and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. Accordingly, we have studied the effect of MG on the formation of free radicals using Electron Spin Resonance (ESR) analysis with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. ESR analysis showed formation of reactive free radicals during exposure of MG to Syrian hamster embryo (SHE) cells. As per mechanism-based toxicology in cancer risk assessment, the chemicals that have the potential to be metabolized to active free radical species could be human cancer hazards. So, we have investigated the effect of MG on the formation of Type II and Type III morphologically transformed foci using SHE cell transformation assay. MG induced dose related transformed foci. Some of these transformed foci were taken out using selective trypsinisation and established immortal cell lines. One of these immortal cell lines was characterized extensively. This immortal cell line showed enhanced DNA synthesis in the form of BrdU incorporation, increased presence of proliferating cell nuclear antigen (PCNA), bcl-2 and p53 proteins by immunohistochemistry. When these immortal cells were injected subcutaneously into nude mice, they developed tumors which were transplantable and histopathologically sarcomas. The present studies indicate that MG could be a potential candidate for two year chemical carcinogenesis rodent bioassays.

CD40-ligation-mediated protection from apoptosis of a Fas-sensitive Hodgkin's-disease-derived cell line.

Modulation of Fas expression and function by CD40 ligation was investigated in the Fas-sensitive human Hodgkin's disease cell line HDLM2. The recombinant human trimeric soluble CD40L (sCD40L) protected this cell line from apoptosis induced by an agonistic Fas antibody at all concentrations tested. sCD40L also protected HDLM2 when added up to 2 h after Fas ligation. Apoptosis induced by a cell-permeable synthetic ceramide could not be prevented by sCD40L. Thus, CD40 ligation is likely to intervene in the early phases of the Fas signal transduction pathway. When CD40 ligation preceded Fas ligation, it rendered the cells refractory to Fas-induced apoptosis. sCD40L-mediated protection could not be attributed to reduction in surface Fas expression, increase in Bcl-2 levels or to increase in the levels of soluble Fas isoforms.

High-resolution mapping of sperm function defects in the t complex fourth inversion.

Structural variants of the mouse Chr 17-specific t complex, known as t haplotypes, express factors that alter the ability of sperm to carry out their roles in the normal fertilization process. In previous studies of males carrying heterospecific combinations of the t complex, we discovered a unique M. spretus/t haplotype phenotype of male sterility. In additional studies with mice carrying a series of M. spretus-M. m. domesticus recombinant Chr 17 homologs and a complete t haplotype (S-+/t), we monitored physiological aspects of sperm function to map a locus (Hst6) responsible for expression of the t-specific "curlicue" sperm flagellar curvature phenotype to 1 cM within the fourth inversion of the t complex. In the present report, we quantitatively analyze the in vitro capability of sperm from mice with similar S-+/t Chr 17 genotypes to fertilize zona pellucida-free mouse eggs. The results identify a locus, Stop1, mapping distal to Pim1, with acute effects on the ability of sperm to penetrate the oolemma. The data suggest that Stop1 is a complex locus consisting of at least two genetic elements, a proximal one overlapping the Hst6 locus, and another, distal to the Hst6 locus. Further quantitative analyses of the "curlicue" phenotype produced by sperm derived from these same animals indicate that expression of this chronic flagellar curvature phenotype also derives from at least two elements, both mapping within the Hst6 locus. Thus, these studies provide higher resolution mapping of the molecular basis of t haplotype-specific sperm dysfunction emanating from In(17)4.

Malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by malachite green: transformation is associated with abrogation of G2/M checkpoint control.

Malachite green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells by MG. In this study, we have studied the effects of MG on cell cycle phase distribution of normal and MG transformed Syrian hamster embryo cells in asynchronous and synchronous cell population. DNA flow cytometric analysis indicated that culturing cells for 48 h in medium containing MG at different concentrations induced dose-dependent G2/M arrest in normal cells. Malignantly transformed cells showed no such dose-responsive accumulation of cells at the G2/M phase of the cell cycle in response to MG. Synchronization studies indicated that in the control, both in the presence and absence of MG, cells followed a normal cell cycle pattern up to 16 h. After 16 h in the absence of MG, cells continued a normal cell cycle, whereas in the presence of MG they accumulated at G2/M phase of the cell cycle. This pattern of accumulation of cells at the G2/M checkpoint control was not observed in either untreated or MG-treated transformed cells. The present study indicates efficient operation of G2/M checkpoint control in control SHE cells and its abrogation in transformed SHE cells.

Oestrogen increases S-phase fraction and oestrogen and progesterone receptors in human cervical cancer in vivo.

Although cancer of the cervix is traditionally considered not to be responsive to steroid hormones, an in vitro study has reported that the addition of oestrogen increased cellular proliferation in a cervix cancer cell line that was inhibited by progesterone. We investigated whether the reported in vitro effects of oestrogen and progesterone on cellular proliferation can be replicated in locally advanced cervical cancer in vivo and whether these effects, if any, are related to oestrogen and progesterone receptor (ER and PgR) content of the tumour. One hundred post-menopausal patients with locally advanced cervical cancer were systematically allocated by rotation to the four treatment groups: (1) control group receiving no treatment; (2) ethinyl oestradiol 50 micrograms: (3) norethisterone 5 mg: (4) a combination of ethinyl oestradiol and norethisterone. Hormone treatment (five doses) was given orally every 12 h. Tissue biopsies were taken before and 12 h after the last hormone treatment. S-phase fraction (SpF) was measured by flow cytometry, and ER and PgR were measured by enzyme immunoassay. Results were analysed using two-factor analysis of variance, the factors being oestrogen-absent or present- and progesterone-absent or present. The main effects of oestrogen were increases in SpF, ER and PgR, which were statistically significant (P = 0.0056, 0.0009 and 0.01 respectively), indicating that there is much greater change in these three parameters in the presence of oestrogen (mean changes 7.808%, 6.258 fmol mg-1 and 12.716 fmol mg-1 for SpF, ER and PgR respectively) than in its absence (mean change -1.986%,-3.041 fmol mg-1 and 1.736 fmol mg-1 respectively). The progestogen main effect and the oestrogen-progestogen interaction were not significant. The rise in SpF, ER and PgR in the presence of oestrogen had a correlation coefficient with the initial ER values of -0.0565, -0.2863 and -0.1230 respectively, none being statistically significant, suggesting that the oestrogen actions were not strictly related to baseline ER concentrations. The combined median baseline ER and PgR values of the four groups were 1.48 fmol mg-1 and 0.80 fmol mg-1 respectively. Our results show that oestrogen is capable of increasing SpF in locally advanced cervical cancer in vivo and may help to revive interest in the use of oestrogen as a radiosensitizing agent in the treatment of this disease.

A comparative study of detection of p53 mutations in human breast cancer by flow cytometry, single-strand conformation polymorphism and genomic sequencing.

The accuracy of immunodetection by dual parameter flow cytometry (FCM), polymerase chain reaction-mediated single strand conformation polymorphism (PCR-SSCP) and genomic sequencing to detect p53 mutations were compared. Analysis by the last two techniques was restricted to exons 5-8. Initially, 110 breast tumours were screened for p53 expression by FCM. Seventy (64%) of tumours were immunopositive. Fifteen highly immunopositive and 15 completely immunonegative tumours were selected for further analysis by PCR-SSCP and genomic sequencing. Eleven out of 15 immunopositive tumours were found to have mutation by PCR-SSCP. Genomic sequencing confirmed the presence of mutation in 10 of these 11 immunopositive tumours. Therefore, four immunopositive tumours failed to show mutation by SSCP and five by genomic sequencing. Of the 15 immunonegative tumours, one showed mutation by both PCR-SSCP and genomic sequencing and one tumour has undergone deletion of the p53 gene. Overall, immunoreactivity correlated with both PCR-SSCP and genomic sequencing in 80% of cases (24/30), and there was 96.5% (28/29) concordance between PCR-SSCP and genomic sequencing. We conclude that there is good concordance between mutations detected by PCR-SSCP and genomic sequencing, but immunochemical detection of p53 overexpression is not an absolute indicator of p53 gene mutation.

Prognosis of breast cancer: evidence for interaction between c-erbB-2 overexpression and number of involved axillary lymph nodes.

The prognostic significance of c-erbB-2 oncogene amplification or overexpression in relation to axillary lymph node metastasis is controversial. We investigated this question in 159 cases of operable breast cancer: 56 patients with node negative disease and 103 patients with pathological involvement of axillary lymph nodes. c-erbB-2 overexpression was assessed by immunohistochemistry using a polyclonal antibody raised against a synthetic peptide fragment of the oncoprotein. The overall incidence of c-erbB-2 overexpression was 35%. c-erbB-2 overexpression was significantly related to survival when all patients were considered (P = 0.0124), and also for patients with positive axillary lymph nodes (P = 0.0026). c-erbB-2 overexpression had no influence on survival of node negative patients (P = 0.7972). A multivariate survival analysis using the Cox proportional hazard model revealed that number of involved lymph nodes, c-erbB-2 overexpression, ER status, and tumour size were independently related to prognosis (P = 0.0000, 0.0012, 0.0112, and 0.0204, respectively). When an interaction term was introduced in the Cox model between c-erbB-2 overexpression and number of involved axillary lymph nodes, a statistically highly significant interaction between these two factors was observed (P = 0.0002), suggesting that the expression of prognostic power of c-erbB-2 overactivity is related to the number of involved axillary lymph nodes. The 159 patients were then subdivided into three groups: node negative (-ve) (56); 1-6 node positive (+ve) (55); and > or = 7 node +ve (48). This cutoff criterion gave the most numerically equitable distribution of the 159 patients into three groups. The relative risk of death increased stepwise from 0.86 (95% CI 0.26-2.78) for node negative patients, to 1.95 (95% CI 0.82-4.63) for 1-6 node positive patients, to 2.23 (95% CI 1.15-4.35) for > 7 node positive patients. Our results suggest that the prognostic influence of c-erbB-2 overexpression increases arithmatically with increasing number of involved axillary lymph nodes.

Prognostic significance of DNA index by flowcytometry in acute lymphoblastic leukaemia.

DNA index (DI) is considered an important prognostic factor in acute lymphoblastic leukaemia (ALL). We undertook this study to correlate DI with other presenting features and response to therapy. Of the 30 patients of ALL treated at our hospital and entered in this study, 15 were put on the aggressive MCP (multi center protocol) 841 protocol and equal number on the Alternate protocol. Eighteen achieved complete remission (13/15 on the former protocol and 5/15 on the later). DI was less than 0.8 in 8 (27%) patients, between 0.8 and 1.2 in 18 (60%) and more than 1.2 in 4 patients (13%). These figures are different from those reported in Caucasians. On multivariate regression analysis, the DI significantly correlated with percentage of blasts in peripheral blood (P = 0.0035). There was no correlation with outcome or response to treatment.

Prognostic association of c-erbB-2 oncogene amplification and protein overexpression in human breast cancer using archival tissues. A comparative study.

The prognostic associations of c-erbB-2 gene amplification analysed by dot-blot hybridization and of c-erbB-2 protein overexpression assessed by immunohistochemistry (IHC) were compared in 161 patients with operable breast cancer using formalin fixed paraffin embedded archival tissues. The efficiency of the dot-blot technique to detect c-erbB-2 amplification was first validated by comparing the results of dot-blot with those of Southern blot hybridization in 134 tumour samples and there was an excellent correlation. In the main series of 161 samples, where results of IHC and dot-blot were compared, 35.4% showed c-erbB-2 overexpression and 17.4% showed gene amplification. Tumours showing overexpression of c-erbB-2 protein had a significantly shorter disease-free survival (DSF) and survival(s) compared to tumours showing no overexpression. A multivariate analysis revealed that c-erbB-2 overexpression was independently correlated with poor prognosis. On the other hand, no significant association between c-erbB-2 gene amplification and DFS or S was observed. We conclude that c-erbB-2 protein overexpression assessed by IHC is a superior prognostic indicator in operable breast cancer compared to c-erbB-2 gene amplification analysed by the dot-blot technique.