Haploinsufficiency for tumour suppressor genes: when you don't need to go all the way

Classical tumour suppressor genes are thought to require mutation or loss of both alleles to facilitate tumour progression. However, it has become clear over the last few years that for some genes, haploinsufficiency, which is loss of only one allele, may contribute to carcinogenesis. These effects can either be directly attributable to the reduction in gene dosage or may act in concert with other oncogenic or haploinsufficient events. Here we describe the genes that undergo this phenomenon and discuss possible mechanisms that allow haploinsufficiency to display a phenotype and facilitate the pathogenesis of cancer.     

Heritable hypersensitivity to induced mutagenesis in the progeny of cell populations exposed to UVC (254 nm)

The ability of mutagens to transform benign papillomas to malignancy in the mouse skin model of multistage carcinogenesis [Hennings et al. Nature 303, 67-68 (1983)] suggests that multiple events may underlie carcinogenic progression, and that mutagenic exposures separated by time can act synergistically. Such synergism may result from initial mutagenic exposure which induces heritable sensitivity to subsequent mutagenic exposures. For example, progeny of X-irradiated V79 cells are hypersensitive to subsequent mutation induced by psoralen plus long-wave ultraviolet light, PUVA [Frank and Williams, Science 216, 307-308 (1982)]. In the present studies 100 to 200 surviving clones of short-wave ultraviolet light (UVC) irradiated V79 cells were assayed for mutation at two loci. Cultures derived from these cells were found to be hypermutable at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus following exposure to PUVA, but showed mutant frequencies similar to control cells following UVC challenge at the HGPRT and ATPase loci.     

Methylcholanthrene-induced Sarcoma and Normal Muscle Fibroblasts of the Mouse — a Comparison of their Ultrastructure

Electron microscope observations of cells from methylcholanthrene induced tumours of the mouse and mouse muscle fibroblasts, grown in vitro , show five main differences in ultrastructure. The surface of the normal fibroblast is smooth when compared with that of the tumour cell; the leading lamella of the normal fibroblast has fewer ruffles (lamellipodia) and fewer cell-substrate plaques than the tumour cell; the tumour cell does not have a layer of cortical filaments like the normal cell but has well marked filamentous tracts in the main body of the cytoplasm and the endoplasmic reticulum of the normal cell is far less abundant and wellorganized than that of the malignant cell. The fact that all these differences are possibly related to the cell surface may be of significance when considering the changes brought about during carcinogenesis.     

Risks of cancer associated with long-term exposure to PUVA in humans: current status--1991.

Since 1975 oral 8-methoxypsoralen administered in association with ultraviolet-A radiation (UVA), (PUVA) has been widely used to treat psoriasis and other cutaneous diseases. PUVA is mutagenic, and in animals carcinogenic. Prospective study of a cohort of patients with psoriasis who were first treated with PUVA in 1975-1976 has provided data on the carcinogenic risk of this treatment. There is a dose-dependent increase in the risk of squamous cell cancer of the skin associated with exposure to PUVA. A recent large-scale Swedish study confirmed this association. The risk of squamous cell cancer of the genitals of males exposed to high doses of PUVA is especially high. A consistent, confirmed, and significant relationship of exposure to PUVA to other types of malignancies in man has not been established. Although highly effective in the treatment of psoriasis, the risk of squamous cell cancer associated with long-term therapy with PUVA must be considered in determining when this therapy is appropriate for an individual patient. Additional study of PUVA-treated patients will better define the full spectrum of the carcinogenic risk of PUVA therapy and the clinical behavior of tumors that arise in association with this treatment.     

Challenge and promise: roles for clusterin in pathogenesis, progression and therapy of cancer

Clusterin (CLU) has been implicated in various cell functions involved in carcinogenesis and tumour progression. There are two known CLU protein isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is proapoptotic, and a secretory form (sCLU) is prosurvival. CLU expression has been associated with tumorigenesis of various malignancies, including tumours of prostate, colon, and breast. Furthermore, CLU expression is modulated by many factors that are believed to regulate tumour growth and/or apoptosis, including 1,25-dihydroxyvitamin D3, transforming growth factor beta-1, ultraviolet radiation, and IR. sCLU upregulation appears to be a general molecular stress response. Presently, preliminary results indicate that therapeutic modalities targeting CLU may be effective in cancer treatment. However, such strategies should make sure that nCLU is not eliminated or reduced. This review summarizes our present understanding of the importance of CLU in various physiological functions including tumour growth, and discusses its relevance to future cancer therapy.     

Insight of nitric oxide signaling: A potential biomarker with multifaceted complex mechanism in colorectal carcinogenesis

Colorectal carcinogenesis (CRC) is the most important health concerns throughout the World as the tumour cells rapidly spread and abruptly grow in colon and rectum to further organs. Several etiological factors are associated with colorectal carcinogenesis. During invasion and proliferation of tumour cells, various mechanistic molecular pathways are involved in the cells. Nitric Oxide pathway (NO) is one of the important cellular mechanisms associated with tumour cells initiation, invasion and progression. Epidemiological evidences suggest that NO has potential role in development of cancer. The multidisciplinary action of NO on the initiation of cancer depends on several factors including cell type, metastasis stage, and organs involved. This review emphasizes the biological significance of NO in each step of cancer metastasis, its controversial effects for carcinogenesis including initiation, invasion and progression.     

p53 and Fas ligand are required for psoralen and UVA-induced apoptosis in mouse epidermal cells

A combination of 8-methoxypsoralen (8-MOP) and ultraviolet-A (UVA) radiation (320-400 nm) (PUVA) is widely used in the treatment of psoriasis and other skin diseases. PUVA is highly effective in eliminating hyperproliferative cells in the epidermis, but its mechanism of action has not been fully elucidated. In this study, we used immortalized JB6 mouse epidermal cells, p53(-/-), and Fas ligand deficient (gld) mice to investigate the molecular mechanism by which PUVA induces cell death. The results indicate that PUVA treatment induces apoptosis in JB6 cells. In addition, PUVA treatment of JB6 cells results in p53 stabilization, phosphorylation, and nuclear localization as well as induction of p21(Waf/Cip1) and caspase-3 activity. In vivo studies reveal that PUVA treatment induces significantly less apoptosis in the epidermis of p53(-/-) mice compared to p53(+/+) mice. Furthermore, FasL-deficient (gld) mice are completely resistant to PUVA-induced apoptosis compared to wild-type mice. These results indicate that PUVA treatment induces apoptosis in mouse epidermal cells in vitro and in vivo and that p53 and Fas/Fas ligand interactions are required for this process, at least in vivo. This implies that similar mechanisms may be involved in the elimination of psoriatic keratinocytes from human skin following PUVA therapy.     

Role of nitric oxide in tumour progression with special reference to a murine breast cancer model

Nitric oxide (NO) is a potent bioactive molecule produced in the presence of NO synthase (NOS) enzymes, which mediates numerous physiological functions under constitutive conditions. Sustained overproduction of NO (and NO-reaction products), typically under inductive conditions, can lead to cell cycle arrest and cellular apoptosis. Furthermore, carcinogenesis may result from mutational events following NO-mediated DNA damage and hindrance to DNA repair (e.g., mutation of tumour-suppressor gene p53). In a majority of human and experimental tumours, tumour-derived NO appears to stimulate tumour progression; however, for a minority of tumours, the opposite has been reported. This apparent discrepancy may be explained by differential susceptibility of tumour cells to NO-mediated cytostasis or apoptosis, and the emergence of NO-resistant and NO-dependent clones. NO-resistance may be mediated by p53 inactivation, and upregulation of cyclo-oxygenase-2 and heat shock protein 70 (HSP70). In a murine mammary tumour model, tumour-derived NO promoted tumour growth and metastasis by enhancing invasive, angiogenic, and migratory capacities of tumour cells. Invasion stimulation followed the altered balance of matrix metalloproteases and their inhibitors; migration stimulation followed activation of guanylate cyclase and MAP kinase pathways. Selective NOS inhibitors may have a therapeutic role in certain cancers.     

Cyclopamine inhibition of the sonic hedgehog pathway in the stomach requires concomitant acid inhibition

The Shh pathway has been implicated in gastric carcinogenesis, and inhibition of this pathway has been shown to inhibit tumour growth in gastric cell lines. Assessing the in vivo efficacy of Shh pathway antagonists in blocking Shh signaling in the stomach is important for clinical trial design, but has not been previously investigated. We investigated the in vivo efficacy of a Shh antagonist, cyclopamine, in correlation to the secondary effects induced by this treatment on gastrin levels and acid secretion. Gastrin has been shown to induce Shh production, processing and activity, which is believed to be mediated by acid secretion. We tested this hypothesis and showed that hypergastrinaemia induces Shh production in vivo, and confirmed that this effect on Shh is mediated by acid secretion. We showed that cyclopamine treatment induces both hypergastrinaemia and Shh, and does not inhibit Gli-1. Inhibition of the effect of hypergastrinaemia on the Shh pathway, in cyclopamine-treated mice, was demonstrated by use of lansoprazole which concomitantly inhibited Gli-1, and did not increase Shh production. Therefore, this evidence suggests that hypergastrinaemia, via increased acid secretion, may increase expression of Shh and that Shh antagonists may require concomitant acid inhibition to successfully inhibit a pathway known to be up-regulated in gastric carcinogenesis.     

Crosstalk between cancer‐associated fibroblasts and immune cells in cancer

Multiple studies have shown that cancer‐associated fibroblasts (CAFs) play an important role in tumour progression, including carcinogenesis, invasion, metastasis and the chemoresistance of cancer cells. Immune cells, including macrophages, natural killer cells, dendritic cells and T cells, play a dual role in the tumour microenvironment. Although increasing research has focused on studying interactions between distinct cells in the tumour microenvironment, the complex relationships between CAFs and immune cells remain unclear and need further study. Here, we summarize our current understanding of crosstalk between CAFs and immune cells, which may help clarify their diagnostic and therapeutic value in tumour progression.     

Carcinogenesis: Mechanism and Criteria of Carcinogenic Activity

The pattern of carcinogenic action is described for locally acting as well as for remotely acting carcinogens. Whether applied locally, injected subcutaneously or given by mouth, aromatic polycyclic hydrocarbons are potentially carcinogenic for all tissues, whereas other known locally acting carcinogens have no such wide action. Solubility and diffusibility of the compound should be considered, but the question of localization of induced tumours is a problem of dose-response relationship.The method for evaluating carcinogenic action of a compound is based on the readiness with which the tumour is induced, and not in terms of the intensity of the response. The average latent period and the percentage of tumour yield are the two measures used. Since, at the present time, no chemical tests are available to determine whether a substance is carcinogenic, one must resort to biological methods of testing carcinogenic activity.The value of a long-term test for carcinogenesis, under a given set of rigidly controlled conditions, is emphasized, since there are so many variables which singly or in combination may alter the final effect of a given substance.     

Growth factors and oncogenes in breast cancer

The growth of normal breast epithelial cells is regulated by a complex interacting system of polypeptide factors and by steroid hormones. The cells respond to these factors through receptors which generate mitogenic and other intracellular signals. These second messengers provoke complex responses which may ultimately result in DNA replication and cell division.A comparison of normal cells and tumour cells, either in culture or from primary tumour biopsies, has revealed differences in growth factor and growth factor receptor expression. Such changes may represent aspects of the process of malignant transformation. In addition some evidence suggests that changes in second messenger systems may also occur. Finally several changes in nuclear oncogenes have been observed in breast cancers.It has been proposed that changes in the nuclear oncogenes, perhaps involving the loss of function of tumour suppressor genes, may allow cells to enter the cell cycle. Changes in growth factors, their receptors or intracellular second messenger systems may stimulate unregulated growth. The combination of these events provide a model for the process of carcinogenesis.     

Cancer-associated genes can affect somatic intrachromosomal recombination early in carcinogenesis

The pKZ1 recombination mutagenesis model has provided a sensitive assay where we study somatic intrachromosomal recombination (SICR) as a mutation end-point. SICR is associated with non-homologous end-joining repair of double-strand breaks and can result in chromosomal inversions and deletions, both of which are common chromosomal aberrations identified in cancers. It has been difficult to study the effect of cancer-associated genes on chromosomal changes prior to tumour formation in vivo because of a lack of appropriate test systems. We hypothesised that cancer-associated genes play a role in formation of chromosomal aberrations and that the pKZ1 model would provide a system in which such a role could be studied in the initial steps of carcinogenesis. Transgenic tumour model mice were bred to pKZ1 mice to produce double transgenic animals. SICR inversion events were scored in mouse tissues at an early time, prior to evident tumour formation, and compared with endogenous pKZ1 SICR levels. Over-expression of the c-myc proto-oncogene resulted in a significant 2.1-fold increase in SICR in spleen. Loss of Msh2 and expression of the SV40 T antigen resulted in a significantly reduced SICR frequency (0.3 of the endogenous frequency in pKZ1 mice) in spleen and prostate respectively. Therefore SICR was affected in the case of all three cancer-associated genes studied. We hypothesise that the increase and decrease in SICR in the presence of cancer-associated genes results from incorrect repairing of double-strand breaks. The data presented here suggest that the pKZ1 model may provide a powerful tool for studying the effect of cancer-associated genes on chromosomal changes in the early stages of carcinogenesis.     

MITOTIC CONTROL IN ADULT MAMMALIAN TISSUES

Mitotic homeostasis: Mitotic control is maintained by the interaction of a tissue-specific mitosis-inhibiting chalone, which permeates the whole tissue, and a non-tissue-specific mitosis-promoting mesenchymal factor, which originates in the connective tissue and acts only on connective-tissue-adjacent cells. In the basal layer of the epidermis the mitotic rate is determined by the relative concentrations of these two substances; in the distal layers the chalone is dominant so that all cells must become post-mitotic, age, and die. Thus the perfect balance between cell gain and cell loss that is maintained equally in hypoplasia, normality, and hyperplasia is ensured by the fact that all cells forced distally by mitotic pressure enter a chalone concentration that is high enough to direct them into post-mitosis and so to their deaths. The mitotic rate of the basal epidermal cells and the ageing rate of the distal cells are both inversely related to the chalone concentration. A change in the mitotic rate is matched by an equal change in the ageing rate so that, within limits, epidermal thickness (or mass) remains constant. Epidermal thickness is determined by the tissue-specific ratio, mitotic rate: ageing rate; it is influenced by the mitotic rate only when this exceeds a certain critical level. Evidently all epithelial tissues, even when these form solid masses (e.g. liver hepato-cytes), have a similar control mechanism, the ‘basal cells’ being those that are connective-tissue-adjacent and the ‘distal cells' those that are not. Tissues that are not connective-tissue-based (e.g. erythrocytes and granulocytes) have specialized mechanisms involving differentiation from relatively undifferentiated stem cell populations, as also do the connective tissues themselves. Local tissue damage leads via local chalone loss to a temporarily and locally increased mitotic rate; chronic damage leads via chronic chalone loss to hyperplasia, the increase in tissue mass being limited by the reduced life-span of the post-mitotic cells. Compensatory hypertrophy When a tissue mass is so large (e.g. the hepatocytes) in relation to the total body mass that the escaping chalone forms a significant systemic concentration, extensive damage leads to compensatory hypertrophy. The reduced tissue mass (e.g. after partial hepatectomy) produces less chalone, leading to a reduced systemic concentration, and therefore a higher chalone loss from the surviving tissue. This results in a general mitotic response in that tissue, as the relative power of the mesenchymal factor increases, and thus to an increase in tissue mass. Growth ceases when the normal tissue mass is attained. When a large tissue suffers chronic damage (e.g. liver cirrhosis) the chronic chalone lack results in hypertrophy, which is limited by the reduced life-span of the post-mitotic cells. Tumour growth Mitotic control is lost when the chalone concentration falls so low that the ‘distal cells’ remain mitotic; cell gain then exceeds cell loss and a tumour appears. Such chalone loss is related to permanent membrane damage, which may be the central event in carcinogenesis. The evidence is that a tumour continues to produce and to respond to the chalone of its tissue of origin. As a tumour grows the systemic concentration of its chalone rises steadily so that there is an increasing mitotic inhibition, first, in the parent tissue, and second, in the tumour itself. Thus tumour growth may be described as an exponential process limited by an exponential retardation. This means that, if the host survives, the tumour growth will cease and the tumour mass will reach a plateau. This is a negative feedback mechanism which differs from compensatory hypertrophy only in that, at the plateau, the mass attained is greater than normal, and also in that, at any time, further cell damage may cause the tumour to ‘progress’. When this happens the new and higher plateau may be unattainable before the host is killed. Tumour growth is normally slower than would be expected if the mitotic advantage were the only factor involved; clearly tumour growth is usually inhibited by factors other than the chalone, in particular perhaps by the immune response to the altered cell membrane. It is an especial pleasure to acknowledge the constant help and encouragement that has been given by Johanna U. R. Deol.     

Mechanisms and effectors of MIF-dependent promotion of tumourigenesis

The importance of secreted cytokines and growth factors in the development and promotion of malignancies is often underestimated. Many different soluble, extracellular gene products participate in processes that collectively contribute to the growth and survival of a developing neoplasm. These secreted molecules can, directly or indirectly, play a central role in uncontrolled tumour cell division, angiogenic stimulation or suppression of tumour cell immune surveillance. One of the first cytokine activities ever described, macrophage migration inhibitory factor (MIF), is unique to these soluble mediators in that it participates in all of these pro-tumourigenic processes. Overexpressed in most tumour types examined, MIF has been shown to promote malignant cell transformation, inhibit tumour cell-specific immune cytolytic responses and strongly enhance neovascularization. Despite this broad array of activities, the elucidation of molecular and cellular mechanisms involved in MIF-dependent bioactions has remained elusive. This review will focus on recently characterized phenotypes and mechanistic effectors thought to be associated with MIF-dependent promotion of neoplastic processes and discuss their relative importance in carcinogenesis.     

Antiapoptotic role of p38 mitogen activated protein kinase in Jurkat T cells and normal human T lymphocytes treated with 8-methoxypsoralen and ultraviolet-A radiation

A combination of 8-methoxypsoralen and ultraviolet-A radiation (320–400 nm) (PUVA) is used for the treatment of T cell-mediated disorders, including chronic graft-versus-host disease, autoimmune disorders, and cutaneous T-cell lymphomas. The mechanisms of action of this therapy, referred to as extracorporeal phototherapy, have not been fully elucidated. PUVA is known to induce apoptosis in T lymphocytes collected by apheresis, however no information is available concerning the underlying signaling pathways which are activated by PUVA. In this study, we found that PUVA treatment of Jurkat cells and human T lymphocytes up-regulates the p38 MAPK pathway but not the p42/44 MAPK or the SAPK/JNK signaling networks. The use of a pharmacological inhibitor selective for the p38 MAPK pathway, SB203580, allowed us to demonstrate that this network exerts an antiapoptotic effect in PUVA-treated Jurkat cells and T lymphocytes from healthy donors. Moreover, the effect of SB203580 was not due to a down-regulation of the Akt survival pathway which was not activated in response to PUVA. These results may suggest that p38 MAPK-dependent signaling is very important for the regulation of survival genes after exposure to PUVA. Since the therapeutic effect of PUVA seems to depend, at least in part, on apoptosis, further studies on the apoptosis signaling networks activated by this treatment might lead to the use of signal transduction modulators in combination with PUVA, to increase the efficacy of this form of therapy.     

Connexins and cancer

The hypothesis, that gap junctional intercellular communication plays a key role in carcinogenesis and more generally in growth control was formulated nearly 40 years ago. From this time, data accumulated, showing that this type of communication is frequently decreased or absent in cells treated with tumor promoting agents, among transformed cells or between transformed/tumor cells and normal cells. This observation has been made on various cell types and whatever their tissue and species origins, by using in vitro and in vivo models. It led to the general assumption that the inhibition of gap junctional intercellular communication may play a role in carcinogenesis at two levels: (1) during tumor promotion by favoring the clonal expansion of initiated cells and (2) after the phenotypic transformation of cells by preventing the diffusion of putative "normalizing" factors between tumor cells and surrounding normal cells. During the past decade, the discovery that gap junction proteins, the connexins (Cx), may act as tumour suppressors, by reverting the phenotype of transformed cells confirmed the idea that their lack of function would be actively involved in carcinogenesis. However, we still do not know precisely what are the molecular processes that gap junctional intercellular communication may regulate and still do have very few data concerning the gap junction situation in human cancers. All these aspects are presented from an historical point of view and discussed below.     

Cytochemical and biochemical microanalysis of carcinogenesis

Conclusions While the understanding of early cellular changes gleaned from conventional histopathology alone has been rather limited, modern cytochemical methods at the light and electron microscope level have revealed in a number of experimental models and in some human tumour types important results which indicate that a sequence of qualitatively different cell populations is followed during carcinogenesis Some of the cytochemical and electron microscope findings can be correlated with specific cytopathological phenomena which are readily detectable in routine H & E sections. Thus the evaluation of animal experiments concerned with the testing of chemical compounds for carcinogenicity has been improved. The use of simple cytochemical techniques may considerably increase the reliability of the results. With respect to the further elucidation of the mechanism of neoplastic cell transformation, cytochemistry has broadened research horizons. The identification of putative preneoplastic and early neoplastic cell populations by cytochemical methods allows for the first time the microdissection and subsequent detailed investigation of target cells of the carcinogen which are at a high risk of becoming cancer cells. The combination of cytochemical and biochemical microanalysis seems to be the most useful tool for clarifying a number of important problems of carcinogenesis at present.Dedicated to Professor Ekkehard Grundmann on the occasion of his sixtieth birthday.     

Absence of the c-Ha-ras and c-Ki-ras oncogene mutations in the hermaphroditic fish Rivulus marmoratus papillary thyroid carcinomas induced by N-methyl-N'-nitro-N-nitrosoguanidine

Previously we reported that papillary thyroid carcinomas were predominantly induced at high frequency by a low dose of N -methyl- N' -nitro- N -nitrosoguanidine (MNNG) in the hermaphroditic fish Rivulus marmoratus . In the current study, polymerase chain reaction (PCR) amplification and direct sequencing were used to examine the point mutations of Ha- and Ki- ras genes, which may be associated with papillary thyroid tumour development in rivulus. Thirty-three tumour samples were tested, however, no mutations were detected in rivulus Ha- and Ki- ras genes. In human and rodent models, it has been reported that ras gene mutations in papillary thyroid tumours occurred preferentially in the N- ras gene in general, while follicular tumours contained activated Ha- or Ki- ras gene mutations. This may explain why papillary thyroid carcinomas in rivulus were not mutated at codon 12, 13 or 61 of exon 1 or exon 2 of the rivulus Ha- or Ki- ras gene. These results imply that another oncogene, such as the N- ras gene and others, may be preferentially activated in rivulus papillary thyroid carcinomas, and also give valuable information for comparative studies of papillary thyroid carcinogenesis.