Proportions of mitotic and apoptotic cells in a range of untreated experimental tumours

The quantitative aspect of apoptosis in experimental tumours is often neglected. In this study, the apoptotic and mitotic indices for a range of tumours have been determined at light microscope level. It has been found that the apoptotic levels fall into a consistent range for all tumour types and agree well with those described by previous workers. It is suggested that these might be basic parameters of tumour expansion, as relevant to growth kinetics as mitotic levels.     

Degree of differentiation and blood vessel proximity in B16 melanoma

Corded structures consisting of rows of viable tumour cells around a central blood vessel are present in a number of transplantable mouse tumours, including transplantable B16 melanomas. These tumours were used to assess, by stereological means at the EM level, the range of differentiation of the melanoma cells, according to their position in relation to the central blood vessel. The mitotic index was also determined for perivascular and peripheral tumour cells separately. Furthermore, the transition of peripherally located cells into necrotic tumour cells is described at the EM level. Results shown an important increase in differentiation in peripheral tumour cells, whereas the mitotic index is highest in perivascular cells. Necrotic peripheral cells show features of apoptotic necrosis, together with necrosis of the ischaemic type. Results indicate that both proliferation and differentiation of melanoma cells are related to their position around a central blood vessel, and that peripheral necrosis is not exclusively due to lack of oxygen.     

High levels of Fas ligand and MHC class II in the absence of CD80 or CD86 expression and a decreased CD4<Superscript>+</Superscript> T cell Infiltration,enables murine skin tumours to progress

It is still not clear why some tumours will be recognized and destroyed by the immune system, and others will persist, grow, and eventually kill the host. It has been hypothesized that tumour cells might evade immunological destruction by expressing Fas ligand (FasL), a molecule which induces apoptosis in Fas(+) target cells. However, the role of FasL in creating an immune privileged status within a tumour remains controversial. To determine whether FasL is associated with skin tumour progression, we developed a tumour model enabling us to compare two squamous cell carcinomas (SCC). One is a regressor SCC which spontaneously regresses after injection into syngeneic mice. The other is a progressor SCC which evades immunological destruction. Detailed flow cytometric analysis was used to study tumour cell expression of FasL, Fas, CD80, CD86 and MHC class II. We also analysed the percentage of apoptotic tumour cells in vivo using annexin V and correlated skin tumour progression with CD4 and CD8 T cell infiltration. Progressor tumours expressed high levels of FasL in vivo, which was virtually absent from regressor tumours. The percentage of progressor tumours expressing MHC II was significantly greater than regressor tumours, while neither tumour expressed CD80 or CD86 costimulatory molecules. Consistent with a regressor phenotype, the percentage of viable tumour cells was significantly lower for regressor compared to progressor tumours which coincided with a significantly larger CD4(+) T cell infiltrate into the tumour mass. The results suggest that progression of skin tumours occurs if tumour cells express high levels of MHC II but not costimulatory molecules such as CD80 or CD86. This implies that tumours may induce anergy in CD4(+) T cells via MHC II antigen presentation in the absence of costimulation. To ensure escape from the immune system, tumours may then kill these T cells via a FasL-dependent mechanism.     

Dietary mistletoe lectin supplementation and reduced growth of a murine non-Hodgkin lymphoma

The growth of a murine non-Hodgkin lymphoma (NHL) tumour has been shown to be reduced by incorporating mistletoe lectin (ML-1) into the diet. The morphological characteristics of NHL tumours in mice fed ML-1-supplemented diets were different from those in LA (control)-fed mice. The degree of mitotic activity was lower and nuclear area reduced. The degree of lymphocyte infiltration was increased in tumours from ML-1 fed mice and this was accompanied by a high incidence of apoptotic bodies. Visual observation of NHL tumours from individuals fed ML-1 diet showed a poorly developed blood supply in contrast to control-fed mice. A major reduction in number of blood capillaries in NHL tumours was confirmed by microscopic evaluation of tumour sections. The results suggested an anti-angiogenic response in ML-1-fed mice. The feeding of ML-1 compared to control diet thus provided several identifiable changes in the morphology of NHL tumours which were consistent with the observed reduction in tumour weight. There was no longer histological evidence of viable tumour in 25% mice fed the ML-1 diet for 11 days. Morphological studies of the small bowel indicated (a) that the lectin induces hyperplasia, and (b) that the lectin binds avidly to lymphoid tissue of Peyer's patches. There was evidence of limited endocytosis of the lectin. An experiment where ML-3 was added to the diet of mice three days after inoculation of tumour cells showed that the lectin was able to slow down further growth of an established tumour. The results show that ML lectins induce powerful anti-cancer effects when provided by the oral route.     

Estrogen withdrawal-induced human breast cancer tumour regression in nude mice is prevented by Bcl-2

We recently showed that estrogen induces expression of the anti-apoptotic protein, Bcl-2 in MCF-7 human breast cancer cells. Since estrogen-dependent breast tumours can regress following estrogen withdrawal, we hypothesized that stable Bcl-2 expression would prevent estrogen-withdrawal induced regression of MCF-7 tumours. We therefore established tumours in ovariectomized female nude mice implanted with an estrogen-release pellet using untransfected MCF-7 cells or MCF-7 cells stably transfected with a Bcl-2 cDNA sense or antisense expression vector. All tumours grew at similar rates indicating that Bcl-2 levels have no effect on tumour formation. After removal of the estrogen pellet, Bcl-2 antisense tumours and untransfected MCF-7 tumours regressed means of 49% and 52%, respectively, after estrogen pellet removal whereas Bcl-2 sense tumours were significantly stabilized. Regressing tumours displayed characteristics of apoptotic cells. These results show that Bcl-2 can prevent hormone-dependent breast tumour regression and are consistent with the notion that decreased Bcl-2 levels following estrogen withdrawal renders hormone-dependent breast tumour cells sensitive to apoptotic regression.     

Macrophages and Apoptotic cells in Human Colorectal Tumours

The numerical relationship between tumour associated macrophages (TAM) and apoptotic cells in 12 human colorectal tumours was evaluated. TAM were labelled immunohistochemically and apoptotic cells were visualized by counterstaining with haematoxylin and eosin (H&E). The stereological techniques. Cavalieri's estimator of volume and the Disector were used to estimate both tumour volume and numerical density of both cell types. The occurrence of TAM per unit volume of tissue increased with increasing tumour volume to a maximum in a tumour of 110·5 cm³, after which numbers declined. Levels of apoptosis also increased with tumour volume though more erratically than levels of TAM and declined for tumour volumes greater than 80 cm³. This is the first report of an attempt to assess the relationship between apoptotic cells and TAM in human tumours.     

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.     

Epidermal chalones and squamous cell carcinomas. The growth inhibitory effects of aqueous epidermal extracts (G1 and G2 chalones) on the epidermis and on a transplantable keratinizing carcinoman in nude mice.

Balb/c/nu nude mice that had been transplanted with a moderately differentiated squamous cell carcinoma were injected i.p. with different doses of epidermal chalone, and control animals were injected with saline. The labelling indices (H3TdR) and the mitotic rate (stathmokinetic method with vinblastine sulphate) were determined. In the untreated animals, both the labelling index and the mitotic rate of the tumor were considerably higher than in the epidermis, and the rate of cell birth was almost twice that of the epidermis. Higher doses of chalone were needed to reduce the labelling index for the tumour than for the epidermis, and there was generally a less pronounced dose/response relationship in the tumours than in the epidermis. The same was true of the mitotic rate but here the results were not as obvious as for the labelling index. A possible explanation of the results may be that the tumour cells are less sensitive than epidermal cells to the injected chalones, or that reduced vascularization of the transplanted tumour may lead to reduced access of chalone, or that tumour necrosis may pay a role. However, it is evident that the tumour cells react less than the epidermis to both the G1 and the G2 chalone, and thus the findings of this study do not provide any evidence against the theory that epidermoid transplanted tumours are less sensitive to epidermal chalones than normal tissue of the same histogenetic origin.     

Vascular endothelial growth factor (VEGF), a survival factor for tumour cells: implications for anti-angiogenic therapy

Angiogenesis is central to both the growth and metastasis of solid tumours. Anti-angiogenic strategies result in blood vessel regression accompanied by tumour cell apoptosis. Radiotherapy and many chemotherapeutic agents kill tumours by inducing apoptotic cell death. We propose that, in addition to its role as an angiogenic factor, vascular endothelial growth factor (VEGF) can act as a survival factor for tumour cells protecting them from apoptosis. Thus anti-angiogenics, in particular those directed against VEGF, have multiple anti-tumour effects. We suggest that anti-VEGF strategies prevent vessel growth and block a tumour cell survival factor, VEGF, rendering tumour cells more sensitive to chemotherapy and radiotherapy. In addition, as chemotherapy and radiotherapy have been shown to increase VEGF expression, anti-VEGF strategies may overcome therapy- induced tumour cell resistance.     

NuMA overexpression in epithelial ovarian cancer

Highly aneuploid tumours are common in epithelial ovarian cancers (EOC). We investigated whether NuMA expression was associated with this phenomenon.NuMA protein levels in normal and tumour tissues, ovarian cell lines and primary cultures of malignant cells derived from ovarian ascitic fluids were analysed by Affymetrix microarray analysis, immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF), with results correlated to associated clinical data. Aneuploidy status in primary cultures was determined by FACS analysis.Affymetrix microarray data indicated that NuMA was overexpressed in tumour tissue, primary cultures and cell lines compared to normal ovarian tissue. IHC revealed low to weak NuMA expression in normal tissues. Expression was upregulated in tumours, with a significant association with disease stage in mucinous EOC subtypes (p = 0.009), lymph node involvement (p = 0.03) and patient age (p = 0.04). Additional discontinuous data analysis revealed that high NuMA levels in tumours decreased with grade (p = 0.02) but increased with disease stage (p = 0.04) in serous EOC. NuMA expression decreased in late disease stage 4 endometrioid EOCs. High NuMA levels decreased with increased tumour invasion in all subtypes (p = 0.03). IF of primary cultures revealed that high NuMA levels at mitotic spindle poles were significantly associated with a decreased proportion of cells in cytokinesis (p = 0.05), increased binucleation (p = 0.021) and multinucleation (p = 0.007), and aneuploidy (p = 0.008).NuMA is highly expressed in EOC tumours and high NuMA levels correlate with increases in mitotic defects and aneuploidy in primary cultures.     

IN VITRO AND IN VIVO LABELLING OF ANIMAL TUMOURS WITH TRITIATED THYMIDINE

The labelling indices obtained by incubating tumour specimens with tritiated thymidine in vitro under hyperbaric oxygen have been compared with those obtained by labelling a matched tumour in vivo. The correspondence between these individual labelling indices is sometimes very poor; however, the average labelling index derived from groups of tumours labelled in the two ways does not differ significantly. There was a large variation from field to field within any tumour, and considerable variation from one tumour to another within each tumour type. The mitotic index was also compared in the matched tumour preparations; the mitotic index in vitro was almost always considerably lower than the values observed in vivo.     

Molecular nature of genetic changes resulting in loss of heterozygosity of chromosome 11 in Wilms' tumours

Summary In this paper we describe the analysis of genetic changes in chromosome 11 in Wilms' tumours. Using a range of probes for regions 11p15, 11p13 and 11q we have screened DNA from 14 Wilms' tumours together with control DNA obtained from the patients' lymphocytes and their parents. We have been able to demonstrate loss of heterozygosity in 5 of the 14 different Wilms' tumours. In three of these five tumours, loss of heterozygosity did not involve markers for 11p13, 11p15.4 or the proximal region of 11p15.5, but only some markers assigned to the most distal part of 11p15.5. In two of these tumours we could demonstrate unequal mitotic recombination in 11p with breakpoints in the hypervariable regions 5 of the insulin gene and/or 3 of the HRASI protooncogene. In one tumour, from a Beckwith-Wiedemann patient, all markers for the region 11a13-pter became hemizygous; the region 11q13-qter remained heterozygous. These results demonstrate that loss of heterozygosity in Wilms' tumours may not necessarily involve the proposed Wilms' tumour locus at 11p13 but may be limited to 11p15.5. This suggests that not only the 11p13 region, but also the 11p15.5 region is involved in Wilms' tumour development. The possible role of both regions in the development of Wilms' tumour is discussed.     

The expression of oncogenes in tumour tissues of breast carcinoma patients

The balance between Bcl-2 and c-Myc and c-Jun seems to be an important determinant of cellular sensitivity to the induction of apoptosis. High expression of Bcl-2 was noticed to be strongly related to low rates of apoptotic cell death. The mean value of the apoptotic index was 45.0% in Bcl-2-negative tumours and 7.5% in Bcl-2-positive tumours. C-Myc and c-Jun accumulation were associated with the absence of Bcl-2 expression and with increased apoptotic activity. The loss of Bd-2 expression was strongly correlated with increased apoptotic cell death. The inverse correlation is between apoptotic and mitotic index. A high mitotic index exists in most patients with a low apoptotic index. Bcl-2, c-Myc and c-Jun does not only take part in cell death, but also in cell division in breast carcinoma cells in which the regulation of cell division and cell death are strictly connected.     

The Role of Mitochondria in Glioma Pathophysiology

It has long been recognised that malignant tumours favour aerobic glycolysis to generate ATP and contain abnormalities of the intrinsic, mitochondria-dependent, apoptotic pathway, suggesting the involvement of dysfunctional mitochondria in tumour pathophysiology. However, the mechanisms underlying such processes in gliomas are poorly understood. Few recent studies have evaluated mitochondrial ultrastructure and proteomics in the pathophysiology of malignant gliomas. However, aberrant energy metabolism has been reported in gliomas and mitochondrial dysfunction links to glioma apoptotic signalling have been observed. Mitochondrial structural abnormalities and dysfunction in malignant gliomas is a neglected area of research. Definition of abnormalities in mitochondrial proteomics, membrane potential regulation, energy metabolism and intrinsic apoptotic pathway signalling in gliomas may open novel therapeutic opportunities.     

Synaptonemal complex protein SYCP3 impairs mitotic recombination by interfering with BRCA2

The meiosis-specific synaptonemal complex protein SYCP3 has been reported to be aberrantly expressed in tumours. However, in contrast to its well-defined function in meiosis, its possible role in mitotic cells is entirely unknown. Here, we show that SYCP3 is expressed in a range of primary tumours and that it impairs chromosomal integrity in mitotic cells. Expression of SYCP3 inhibits the homologous recombination (HR) pathway mediated by RAD51, inducing hypersensitivity to DNA-damaging agents such as a poly(ADP-ribose) polymerase (PARP) inhibitor and chromosomal instability. SYCP3 forms a complex with BRCA2 and inhibits its role in HR. These findings highlight a new mechanism for chromosomal instability in cancer and extend the range of PARP-inhibitor sensitive tumours to those expressing SYCP3.     

Changes in cellularity induced by radiation in a solid tumour.

The growth, and cellular responses of Morris hepatoma 3924 A to a locally-administered dose of 3750 R X-rays were studied using the following parameters; (1) relative tumour volume changes; (2) tritiated thymidine (3H-TdR) incorporation into DNA; (3) tumour DNA content and (4) cellular analysis, including 3H-TdR labelling index, mitotic index, aberrant mitotic frequency and relative cell density. Before depression of tumour growth, cell proliferation is temporarily interuppted. As proliferation is reinitiated, a short-lived synhcrony and prolongation of cell-cycle traverse are reflected in (a) the labelling index and mitotic index, (b) the relative cell density, and (c) the rate of incorporation of 3H-TdR into DNA. Within 4 days after radiation, cell proliferation and 3H-TdR incorporation are significantly depressed. Simultaneously there are reductions in both the relative cell density and tumour DNA contents, and these remain depressed as the tumours initiate regression. From these studies, it is apparent that the cellular responses to radiation insult occur well in advance of measurable volume changes and are observed both in tumours that continue to regress and in those that initiate regrowth.     

The tumour suppressor RASSF1A regulates mitosis by inhibiting the APC-Cdc20 complex

The tumour suppressor gene RASSF1A is frequently silenced in lung cancer and other sporadic tumours as a result of hypermethylation of a CpG island in its promoter. However, the precise mechanism by which RASSF1A functions in cell cycle regulation and tumour suppression has remained unknown. Here we show that RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression. RASSF1A localizes to microtubules during interphase and to centrosomes and the spindle during mitosis. The overexpression of RASSF1A induced stabilization of mitotic cyclins and mitotic arrest at prometaphase. RASSF1A interacts with Cdc20, an activator of the anaphase-promoting complex (APC), resulting in the inhibition of APC activity. Although RASSF1A does not contribute to either the Mad2-dependent spindle assembly checkpoint or the function of Emi1 (ref. 1), depletion of RASSF1A by RNA interference accelerated the mitotic cyclin degradation and mitotic progression as a result of premature APC activation. It also caused a cell division defect characterized by centrosome abnormalities and multipolar spindles. These findings implicate RASSF1A in the regulation of both APC-Cdc20 activity and mitotic progression.     

Mitotic crossing-over by anticancer drugs in Saccharomyces cerevisiae strain D5

Treatment with an anticancer drug causing mitotic crossing-over could lead to expression of recessive genes, previously masked in a heterozygote. Used clinically, such drugs might cause an increased risk of cancer in cases of familial tumours, such as Wilm's tumour or retinoblastoma. Potentially, novel forms of drug resistance could also be unmasked by such a recombinogenic event. We have estimated the extent of this potential problem in current clinical drugs by comparing a range of antitumour agents for ability to cause mitotic crossing-over in Saccharomyces cerevisiae strain D5. We have compared these data with ability to cause an increase in total aberrant colonies in the same experiments. Although many of the agents known to cause point mutation also have some ability for mitotic crossing-over, there are also point mutagens which have little recombinogenic potential. Conversely, some effective recombinogens appear to be either very specific or rather ineffective point mutagens. Although the most generally effective agents in the present experiments were alkylating agents, several other types of drug including DNA-cutting agents, topoisomerase inhibitors, other DNA-binding drugs and antimetabolites may stimulate mitotic crossing-over. None of the mitotic inhibitors or the DNA minor groove binding drugs tested caused recombinogenic events. It would seem that the ability to induce mitotic crossing-over is an important endpoint in its own right. Assays for this event might provide an important complement to other assays commonly required for registration of new pharmaceuticals.