2.1.1. Physical factors

Different types of non-ionizing and ionizing radiations are majorly involved in the mechanisms of carcinogenesis.

Non-ionizing radiations are electromagnetic, with low penetration, and present a real danger for eyes and skin. This group includes ultraviolet radiation, light radiation and infrared radiation, all having the sun as the main source.

Ultraviolet B rays are mutagenic and carcinogenic, but they have a weak penetrability of only 10%, being stopped by the horny layer. They are absorbed by the ozone layer. Ultraviolet A rays penetrate the dermis easily.

Albinism and Xerodermia pigmentosum are hereditary factors predisposing to actinic cancer. Experimental actinic cancers are sarcoma like cancers. The main spontaneous actinic cancers in domestic animals are: vulvar melanoma in Angora goats; conjunctival squamous cell carcinoma in Hereford and Norman cattle; squamous cell carcinoma in Scottish Shepherds; squamous cell carcinoma of the external ear in white cats; squamous cell carcinoma of the external ear in sheep.

Directly ionizing radiations are electrically charged particles: negative charge, electrons, such as beta rays; positive charge, alpha particles.

Indirectly ionizing radiations are particles without electric charges: photons, X rays and Gamma rays.

The biological action of the ionizing rays depends on numerous factors: doses distributed in time add their effects, acting through summation; they can act by a single dose; their action is influenced by the presence or absence of radiosensitizing substances, such as oxygen, and radioprotective substances; the species can be more sensitive or on the contrary, resistant. For each species the following will be taken into account: the water and oxygen content; the mitotic index and the degree of tissue differentiation.

Regarding the biological action, the major impact is at the level of the cell nucleus, chromosomes (ruptures, deletions, translocations) and DNA (thymine peroxidation, rupture of a phosphorylated bond at the level of a chain and breakage of one or two filaments). The cytoplasm is another target site, causing: arrest of metabolism after the destruction of enzymatic molecules, alteration of mitochondrial and lysosomal membranes, with the release of the enzymatic content.

These biological actions result in either the death or the survival of the cell as carrier of a mutation.

In human pathology, radioleukemia and radiodermitis that induce squamous cell carcinomas in radiologists are well known, as well as infant leukemia following fetal radiation, thyroid cancer after cervical radiation, etc.

Experimental local radiation in rats causes radiodermitis evolving into sarcomas; thyroid cancer and hypophyseal tumors.

Cancers induced by radiation are characterized by late onset, after 10 or more years, risk persisting over a period longer than 30 years.

Repeated low intensity traumas can determine metaplastic, dysplastic changes, and even tumor proliferation. The involvement of traumas in the development of tumors should be regarded with circumspection, since exophytic tumors are submitted to the action of traumatic factors that cause erosions and ulcers.

Osteomas occurring at the level of bones on which repeated traumas are exerted are well known in animals.

2.1.2. Chemical factors

The first observation in the natural history of carcinogenesis concerning the intervention of a substance in the appearance of a tumor belongs to Sir PERCIVAL POTT, from 1775. This London doctor understands that there is a relation between soot and the appearance of scrotal cancer in chimney-sweeps. Other observations have been subsequently reported. Thus, in 1874, VOLKSMANN remarks the high incidence of skin cancer in tar workers. REHN (1895) describes bladder cancer in workers who come in contact with aromatic amines.

Experimentally, YAMAGIWA and ICHIKAWA (1915) succeed in inducing ear cancer in rabbits, by repeated tar applications.

Clinical and epidemiological observations have demonstrated the increased frequency of a certain cancer type, in a certain geographical area, within a group or profession, correlated with the presence of toxic chemical agents. These findings have led to the notion of carcinogenic substance.

At present, researchers aim to detect the relations existing between the structure of a substance and the carcinogenic effect, to identify ultimate metabolism before biodegradation, air and food pollution, etc.

According to MAGNOL and ACHACHE (1983), the main carcinogenic substances can be classified as follows: mineral substances, organic substances and mixed substances (tobacco, food carcinogens, etc.).

Chemical carcinogens act either directly, causing mutations, or indirectly, reactivating repressed carcinogens.

Depending on the mechanism of action of the chemical substance, WEISBURGER (1976) distinguishes three classes of chemical carcinogens:

1. Direct action or ultimate carcinogens, whose structure confers them the capacity to induce cancer without a previous metabolic activation in the host organism. This category includes nitrosamines, epoxides, ethylenimines and β-propiolactone.
2. Procarcinogens, group that includes the majority of chemical carcinogens that become active after a previous metabolic activation to ultimate carcinogens. Known procarcinogens: aminozoic colorants, aromatic hydrocarbons, aflatoxins, aromatic amines and urethane.
3. Co-carcinogens, which are chemical substances that cannot induce cancer when they are administered alone, but can enhance the carcinogenic effect of other substances. In general, co-carcinogens act as promoters in tissues in which the initiation stage has appeared. Through the reticulo-endoplasmic system, the detoxification of the organism from foreign substances usually occurs by hydrolysis, reduction, conjugation and oxidation, with renal elimination.

Chemical carcinogenic substances are first activated through the hepatic microsomal system, then they bind in a covalent form to the macromolecules (DNA, RNA, proteins) from the target organs.

2.1.3. Biological factors

Numerous carcinogenic substances, either natural compounds or pollutants, are found in human and animal food. Over 80% of carcinogenic substances are found in the environment and are taken by living beings with air, water and food.

There are epidemiological data that prove the role of dietary factors in the etiology of human and animal cancers. An increased number of natural carcinogens are found in human and animal food, either as natural components or contaminants. Natural carcinogens have been identified in plants, microorganisms and animals.

2.1.4. Animal carcinogens

Cantharidin is the active principle of the gross drug called cantharides from Cantharis vesicatoria, Myleabris cichorii and other insects from the Meloidae, Omeridae and Staphilinidae families, having rubefacient and vesicant effects on the skin and mucosae.

In nude mice, papillomas, squamous cell carcinomas, reticular carcinomas and/or malignant lymphomas have been induced by skin painting.

Cantharidin is a weak but complete carcinogen for the skin and the reticuloendothelial system of the mouse; it promotes skin tumorigenesis in mice, which is initiated by other carcinogens.

Quinones (1,4-benzoquinone and its alkyl derivatives), produced by insects from the Tenebrionidae and Opilionidae families, induce skin tumors and pulmonary neoplasms in mice, by subcutaneous injection, by inhalation, respectively.

Certain parasites, especially trematodes, are suspected of having a carcinogenic role in parasitized humans and animals. This is associated with the high prevalence of gallbladder, liver cancer and other neoplasms, in the geographic areas where schistosomiasis is endemic. The carcinogenesis mechanism is not elucidated in this case, since the involvement of schistosoma toxins has not been experimentally verified. Experimental data have demonstrated the presence in the liver, serum or urine of animals with schistosomas, of high enzymatic levels, which can transform procarcinogens or promutagens into reactive metabolic products in certain host organs [77].

The parasites proved to be involved in tumor proliferations and mentioned by the literature are Schistosoma haematobium, which induces bladder cancer in humans, and Spirocerca lupi, which induces esophageal cancer in the canine species. In rats, the Gyngyloma neoplasticum nematode can cause under certain conditions gastric and tongue cancer.

These parasites seem to induce tumors in both man and animals, by their irritating and less toxic action. Parasites cause local irritations, fibrous reactions, metaplasias, and finally, malignant changes [80,114].

The genetic mutation theory

As early as 1987, HANSEMANN remarked the analogies between mutation and cancer; both are hereditarily transmitted to the daughter cells. According to this theory, the origin of cancer is due to structural anomalies of the genes that regulate cell growth and differentiation. Genetic changes can be hereditary or they can occur in the course of life under the carcinogenic action of various pathogenic factors. In this sense, the following arguments can be mentioned:

• – the influence of genetic constitution in the appearance of cancer; chromosomal anomalies are frequently associated with malignant tumors. Chromosomal anomalies associated with malignant tumors are known: Down syndrome (trisomy 21) and Klinefelter syndrome (XXY), associated with leukemia; D deletion syndrome (deletion of the long arm of chromosome 13), with retinoblastoma; Philadelphia chromosome (22-9 translocation) and chronic myeloid leukemia; the presence of hereditary cancer – the susceptibility to a number of tumor locations is transmitted as a hereditary dominant character. These hereditary cancers seem to evolve along two stages: an inherited mutation, followed by a mutation acquired in the course of life. This group of tumors includes: Wilms tumors, retinoblastomas, neuroblastomas and very likely mammary, endometrial, colon cancer, and leukemia;
• – the presence of chromosomal anomalies in the cancerous cell has been detected in almost all malignant tumors. Initially, the karyotype of the tumor cell tends to be unstable and variable, but it gradually stabilizes in a highly aneuploid form;
• – the correlation between mutagenicity and carcinogenicity: the more mutagenic an agent, the more likely it is to be carcinogenic; it is the case of ionizing radiations, alkylating agents and polycyclic hydrocarbons.

In addition to these possibilities of genetic mutations by external factors, spontaneous mutations also occur. In the etiology and pathogenesis of some cancers, a determining factor is represented by endogenous processes that can induce phenomena of intrinsic mutagenesis. A mutational cause of malignancy supposes that among the mutations dispersed throughout the genome there are key-genes that modify the properties of cells, allowing them to escape from the homeostatic mechanisms that regulate cell division, by invasion and metastasis.

Spontaneous mutation is the consequence of lesions that must occur frequently enough to overcome the capacity of the cell to repair the damaged DNA. Spontaneous mutations can be the result of errors caused by DNA polymerases, during DNA replication. Spontaneous mutations can have the same cancer inducing potential as those caused by environmental exogenous agents.

Assuming that a single dominant mutation is oncogenic, the spontaneous mutation rate is sufficient to produce millions of cancerous cells in the course of life. Numerous mutations with cancer inducing potential may have no effect, since cell proliferation is controlled by homeostatic mechanisms that govern the growth and behavior of cells. Multiple and sequential mutagenic events are necessary to initiate carcinogenesis. A mutant phenotype can result from genetic mutations, such as DNA-polymerase, with the production of a modified enzyme that is exposed to errors in the catalysis of DNA synthesis. A mutant phenotype explains chromosomal instability which, as it is known, characterizes tumor progression.

Taking into consideration the extremely high number of proteins involved in DNA replication and repair, it may be concluded that the number of potential targets that can generate mutant phenotypes can be higher than the number of oncogenes known.

Potential sources of spontaneous mutations:

• – chemical DNA instability – among covalent changes, depurine is the most frequent; DNA-polymerases – seem to frequently meet abasic positions, which is why the replication and the mismatch incorporations on the opposite side of these loci is a major source of spontaneous mutations;
• – mutagenesis by free oxygen radicals occurs especially through hydroxyl ions that seem to be the most damaging; the free radicals resulting from oxygen metabolization change the RNA, membrane proteins and DNA. DNA lesions occur frequently enough to represent a source of spontaneous mutation;
• – due to errors in DNA replication, mutagenesis takes place through the erroneous replication of nucleotides, which are by definition mutations; the priority of DNA-polymerase in ensuring DNA replication is proved by genetic studies, and the errors caused by DNA-polymerase increase mutagenesis [113].

The acceptance without reserves of spontaneous mutagenesis as an oncogenic factor might lead to the fatalist conclusion that the appearance of numerous cancers is inevitable.

The aberrant differentiation theory maintains that the origin of cancer is secondary to functional disorders of genetic regulation mechanisms, without structural changes, which explains the designation of this theory as epigenetic theory. It has been experimentally demonstrated that tumor cells of different origins introduced in a fetal environment have led to the resumption of the tumor cell differentiation process, with the loss of the malignant phenotype, as a result of the cell-cell contact or through mediators present in the extracellular environment (PITOT et al., 1985 [80]). These experiments suggest that the neoplastic cell phenotype can be considered the consequence of a reversible disturbance in the genetic region and not necessarily a mutation, at least for some tumor processes.

The viral theory of the origin of cancer has stimulated the study of the proliferation mechanisms of normal and tumor cells, leading to the discovery of viral oncogenes, capable of malignant transformation, and their normal homologues, the protooncogenes. Viral oncogenes act in the nucleus, the cytoplasm and at the cell membrane surface. Their action is carried out by: phosphorylation, initiation of DNA synthesis and transcription regulation [57].

According to HUBNER and TODARO (1969), the majority of vertebrates have oncornavirus genomes integrated in their genetic patrimony, which are automatically transmitted to their descendants by means of germ cells. These viral germs remain inactive for a certain time, being under the dependence of regulating genes, and only manifest after the intervention of depressive factors. The authors suggest the existence of two distinct viral genes, the virogene and the oncogene. The virogene is involved in the replication and release of the virus; at the same time, some cellular genes can modulate the activation of the virogene. The oncogene commands the cell transformation process.

Researches of the relation between cancer and virus have led to the discovery of the cellular oncogene. Incorporated in the genome of a “lending” oncornavirus, these genes acquire carcinogenic potential. The result can be the same if the cellular gene is depressed before the action of mutagenic differentiations on latent regulating genes.

The discovery of cellular oncogenes has allowed the unitary theory of carcinogenesis, which explains why extremely different agents, such as viruses, radiations, chemical substances, etc. cause the same cellular changes, with carcinogenic effects.

The cell selection theory considers cancerization as a result of the selection of a cellular population with increasing autonomy and malignancy, which has gradually adapted to environmental conditions. This theory ignores or minimizes the important role of mutations occurring throughout the duration of the neoplastic process.

The cell selection theory, with all its implications in the appearance and evolution of the neoplasm, is developed in the chapter on tumor generalization and metastasizing.

Multistage carcinogenesis theory

This theory seems to be generally accepted. Carcinogenesis is a multistage process, since between the initial carcinogenic stimulus and the final manifestation of cancer there are several stages (HART and TURTURRO, 1988 [80]).

The period between the fixation of a carcinogen to chromosomal DNA and the appearance of a population of neoplastic cells can be divided in the following stages: initiation, promotion and progression [23].

Initiation

This stage starts with the action of the carcinogen on chromosomal DNA, inducing a lesion, which can be repaired or reproduced.

The biological mechanisms of repair are complex, and possibilities of action for short and long repair are known. Thus, a lesion can be identified by a specific endonuclease that dissects the damaged filament, a DNA-polymerase is fixed at the level of the dissection and ensures synthesis using as a model the intact complementary filament, and a polynucleotide-ligase allows the fusion of the repaired segment and the rest of the filament.

Other enzymes can act for the excision of a changed single base: N-glycosylase, AAE (apurinic-acid-endonuclease and apyrimidinic-acid-endonuclease), exonuclease, DNA-polymerase, lygase, etc.

Postreplication repair. During DNA replication, DNA-polymerase “skips” the injured area that cannot be replicated. The integrity of the newly synthesized filament is restored by an exchange of genetic material between homologous filaments. The gap involving the donor filament will be filled by the intervention of a DNA-polymerase able to replicate an intact complementary filament.

In the repair of a cell lesion, the time factor is essential. Thus, if mitosis is delayed, DNA can be repaired; if not, the lesion will be replicated and transmitted to the new cells.

The initiation stage of a cell starts with the impossibility of repairing of the DNA lesion.

The initiation of a cell involves its genetic compatibility with the activation of different carcinogenic factors that can act alone or in association, in an isolated way or along several stages. Genotoxic factors can be of different kinds: viruses, radiations, chemical substances, etc. Carcinogenic factors have, according to BERENBLUM’s scheme (1982), an irreversible genotoxic action; repair processes do not occur.

Initiation confers the cell proliferative capacities that remain potential, latent, without necessarily leading to the promotion stage. Following the action of chemical, physical or biological factors, the initiated cell presents an irreversible alteration of the genetic material and has the potential to develop a neoplastic cell clone. Initiation represents a rapid process, of the order of minutes or hours, and the initiated cell can remain indefinitely in this state, without producing adverse effects and without being recognized by the defense systems of the organism, since it does not manifest phenotypically [76].

Promotion

Chronic genetic alterations of the initiated cell determine the neoplastic transformation and the appearance of cells that are capable of autonomous growth. The promoter (non-carcinogenic or weakly carcinogenic if used alone) is applied several times after the simple administration of an initiating carcinogen. “Complete” carcinogens are also known; these agents, with certain doses and application modes, induce cancer without the need for the subsequent action of a promoter. Promoters act by the alteration of normal growth processes, by mechanisms similar to hormonal or growth factor mechanisms.

Experimental carcinogenesis studies have proved that the promotion stage presents a first reversible phase and a second irreversible phase.

Tumor promotion is to a great extent, perhaps even totally, associated with epigenetic factors that alter, directly or indirectly, the genomic DNA expression [182]. Promotion would consist of events occurring in the genetic program of terminal proliferation and differentiation. The promoted cell does no longer recognize the differentiation signals, which would normally remove it from the replication population. Unlike initiators, promoters do not bind to DNA, their major target being the cell membrane.

Progression is characterized by marked malignancy and the tendency to induce changes that cause the death of the host. Cells in the progression stage are characterized by genetic changes, gene alterations and rearrangements, even karyotype alterations, and the tumor is phenotypically characterized by a rapid proliferation rhythm, invasive and metastasizing properties, with biochemical and morphological changes. The essential characteristic is the extraordinary instability of the karyotype. In the study of tumor progression, 5-ase-cytidine was observed to have variable effects, being able to activate (onco)genes involved in the progression process, and in other cases to activate oncogene suppressor genes (antioncogenes) [13].

Carcinogenesis appears as a multistage process at molecular level, being triggered either by the action of retrovirus oncogenes, which all induce RNA synthesis and cell division, or by the disturbed, abnormal activity of protooncogenes, one cellular oncogenes [141].

Multistage carcinogenesis theory

Studies have shown that there are genetic mechanisms involving hereditary transmissible DNA alterations, as well as epigenetic mechanisms that involve the expression of one or several genes.

Genetic mechanisms have been demonstrated by the presence of hereditary cancer or by the increased incidence of cancer in congenital chromosomal lesions or in disorders characterized by DNA repair deficiencies.

The evolution of a normal cell towards a cancerous cell is a complex process, which involves multiple stages, leading to the appearance of a clone of cells that no longer have the same control possibilities as normal cells.

SPANDIDOS and ANDERSON (1989) mention three main classes of genes:

1. oncogenes, which derive from altered normal genes, protooncogenes, so that they become activated [88];
2. modeling genes, which can predispose to cancer and are submitted to a mutation. In general, there is a group of heterogeneous genes, some of which are involved in the repair of damaged DNA;
3. oncosuppressor genes, also known as: antioncogenes, tumor suppressor genes or emerogenes. The whole group has the property of inhibiting the cancer phenotype. The mode of functioning and identification of oncosuppressor genes is difficult; it seems that their role is to inhibit cell proliferation, not to directly regulate oncogenes [84].

2.3.1. Oncogenes and antioncogenes

In the process of cell transformation, at least two classes of genes can represent critical targets for carcinogenic agents, oncogenes and antioncogenes and, although the mechanism by which genetic changes lead to cell malignancy is not clearly understood, it is obvious that these sets of genes contribute to the appearance of neoplasms [76].

ONCOGENES represent portions of certain genes that are responsible for the neoplastic transformation of the cell. They are cellular genes that under the action of mutagenic factors or viruses change their structure, control and cell division function. These changes can be primary, being expressed at the level of chromosomes as translocations, deletions and duplications [141]. Oncogenes are changed versions of normal genes, called protooncogenes [26]. The variety of structural or regulating changes underlying protooncogenes, in spontaneous or induced tumors, indicates the presence of a direct correlation between the activated gene and the cell phenotype regardless of the mechanism by which that gene has been activated (mutation, transduction, retroviral insertion, amplification). The repeated implication of the same protooncogenes in the development of numerous types of tumors shows the biological unity of cancer. Neoplastic microevolution offers numerous possibilities, even for a single group of oncogenes. The viral myc gene can transform a wide variety of cellular targets. It can induce hematopoietic, mesenchymal and epithelial neoplasias in a susceptible host. The myc cellular oncogene can also contribute to the development or progression of a tumor in all three major tissues in birds and mammals [76].

The appearance of an oncogene is a necessary but not sufficient condition for carcinogenesis [64]. The changed genes work together to produce, maintain and develop the malignancy state; at least two oncogenes and one or more antioncogenes are supposedly involved in this process.

Oncogenes have a dominant character and produce the malignant transformation by their activation, while antioncogenes induce the same effect, either by their absence or by multiple forms that have a recessive character.

Oncogenes have been identified in retroviruses, being the most potent carcinogens known. At least 20 viral genes that cause changes, including the ras, myc and erb B genes, have been isolated and characterized [164]. Oncogenes can be grouped in two main categories [75]: Oncogenes of viral origin, without homologues in the genes of eukaryotes, such as EIA genes in adenoviruses or x and lor genes in HIV viruses, types I and II; Oncogenes present in highly oncogenic or acutely transformed retroviruses, in avian or mammalian cells.

Protooncogenes are normal cellular genes, potentially contributing to the induction or promotion of malignant tumors when their structure or expression is altered [44]. When normal protooncogenes are transformed into potentially tumorigenic oncogenes, they are considered as activated. Their activation can take place in different ways:

1. Transduction. Retroviruses contain RNA as genetic material. In the course of infection, these viruses synthesize a DNA replica in the chain, doubled by RNA, which is integrated into cellular DNA. Sometimes, the recombination between viral DNA and cellular DNA leads to the incorporation of viral DNA sequences in the host DNA. A comparison of viral and cellular sequences shows that the transduced sequence is damaged compared to its normal cellular equivalent, frequently containing point mutations, deletions and substitutions [175].
2. Insertional mutagenesis. Slowly changing retroviruses can integrate into cellular DNA at various sites, but probably not at random. With a very slow frequency, they can subsequently integrate in the cellular protooncogene, causing its transcriptional activation through regulating sequences, by a long terminal repeat of viral DNA.
3. Chromosomal translocation is frequently found in malignant tumors of hematogenous origin. The investigations performed in this direction have proved that protooncogenes situated at the splitting point are activated as a result of translocation (HALUSKA et al., 1987 [166]). Two mechanisms of activation have been indicated. The first mechanism is represented by chronic myelogenic leukemia, in which, in 90% of patients, malignant cells present a translocation (9:22). The splitting position on chromosome 9 is variable and appears in c-abl protooncogenes. On chromosome 22 the splitting points are grouped between 2 introns of the phi gene (HEISTERKAMP et al., 1983 and STAM et al., 1987 [166]). These translocations create a hybrid gene that will lead to the formation of a chimeric phl-abl protein, with pathogenic properties.
   The second type of translocation, which is completely different, is represented by the Burkitt lymphoma, where the myc oncogene on chromosome 8 overlaps one of the immunoglobulin loci, IgH on chromosome 14, IgK on chromosome 2 or IgK on chromosome 22. The constant aspect of all Burkitt lymphomas is that the myc gene on the derived chromosome is constitutively expressed, while that on the normal chromosome 8 is silently transcribed. The activation of the oncogene in this disease is supposed to involve the expression of myc in cells that would be not otherwise expressed.
4. Mutation. From a cellular line of a bladder carcinoma, the first oncogene, H-ras I has been isolated, in which activation occurs by a point mutation. Recent studies have shown that in both cellular lines and cancerous tissue, point mutations are coding sequences in the ras gene. It has been found that 40% of primary colon tumors in humans present structural mutations of the ras gene [29, 73]. Activations of ras gene mutations have been found in mammary carcinoma and human myelodysplasias [165].
5. Amplification. Protooncogenes are amplified in a great number of tumors in humans. Two models have been observed: one under the form of occasional peculiarities in some tumors, the other with a usual incidence in certain tumors. In advanced mammary cancer stages, an amplification of c-myc and erb B or neu oncogenes has been found, associated with an unfavorable prognosis [174]. The amplification of the N-myc gene is constant in the advanced stages of neuroblasts and associated tumors, being associated with internal metastasizing and short survival [31].

The altered cells that exhibit chromosomal lesions can either amplify their affected chromosomal region, by non-disjunction - which results in trisomy, by duplication and translocation, or lose their unaffected homologous segment, by non-disjunction - which results in monosomy or by deletion [141].

Structural oncology, based on nucleotide sequence identity between human and viral genes, has established that the designation of human oncogenes should be homologous to the corresponding viral oncogenes:

• –the myc gene is located on autosome 8; in Burkitt lymphoma it is translocated to autosome 14; the myc oncogene is carried by the virus of an avian leukemia type;
• –the ras H gene is located on the short arm of autosome 11; it is involved as an oncogene in different solid carcinomas; the ras H oncogene is present in the virus of the murine Harvey sarcoma;
• –the abl gene is located on autosome 9 and has been noted in chronic myeloid leukemia, being present in the Philadelphia autosome.

According to PĂIŞ (1995), the initiation of neoplastic development through oncogenes has the following possible mechanisms:

1. stable position changes that the transcriptional control system makes more active;
2. minor changes in the code sequence, as a result of mobilization;
3. changes in the expression of other genes by the new association provided by oncogene transposition.

The protooncogene becomes quantitatively malignant by the overproduction of its product, its activity being intensified as a result of the action of different carcinogenic (and co-carcinogenic) agents that irreversibly convert an otherwise normal cell to malignant growth.

According to the same author, (physical, chemical, viral) non-carcinogenic agents might exert their role by either the production of new hydrolytic enzymes (e.g. viruses) or the destruction of lysosomes with the release of hydrolytic enzymes in the cytoplasm, enzymes that would subsequently penetrate the nucleus and attack the DNA at sites situated in the proximity of oncogenes, activating them and triggering the malignant tranformation of the cell.

Viruses can disturb the control of cell growth and division, by insertional mutagenesis involving the integration of viral DNA in the host cell genome, or by oncogenes (several genetic loci) that are directly or indirectly responsible for the host cell malignization. RNA genome tumor viruses (retroviruses, leukoviruses and oncornaviruses), as well as DNA genome viruses (papovaviruses and adenoviruses) are known. The existence of numerous sites in cellular DNA is supposed, which can incorporate DNA sequences of several transformed viruses.

Oncosuppressor genes have been identified indirectly, and the hereditary character of some cancer forms has led to the hypothesis of the existence of suppressor genes. Thus, if these genes are inactive, cell transformation will occur. Approximately 10% of human cancers can be attributed to an inherited genetic component with a somatic mutation in another cell [101]. The detection of oncosuppressor genes has been possible by the combination of familial studies with cytogenetics and molecular genetics, including genetic molecular analyses.

Certain proteins determine tumor cell growth suppression, these proteins also reducing the tumorigenicity, invasion and metastasis of cancerous cells. The β-growth transforming factor, interferons, interleukin 2 and the tumor necrosis factor indicate the inhibiting properties of the tumor. Different human tumor cells produce inhibiting tumor cell factors. While these factors inhibit the growth of a variety of human tumor cells, they also stimulate the growth of normal cells [166]. The same authors remark the high conservation level of protooncogenes and oncosuppressor genes in eukaryotes, suggesting that these genes serve important functions. The properties and cellular location of the gene products of oncosuppressor genes suggest several ways by which they can function:

1. some oncoproteins are structurally similar to the secreted growth factors, for example the sis oncoprotein is partially homologous to the β-chain of the platelet derived growth factor – PDGF. The activation of oncogenes can result in the production of growth factors by the tumor cell itself;
2. some oncoproteins are membrane receptors of the growth factor. Thus, the erb B oncoprotein is homologous to the epidermal growth factor receptor, and the product of the fms oncogene is homologous to the colony stimulating factor 1 receptor;
3. the ras, src, fps, abl, yes, fes, crk oncoproteins and the polyomavirus medium T antigen gene product are located on the inner side of the cytoplasmic membrane and are considered to be involved in the mediation of receptor signaling. These products coded by oncogenes can act immediately downstream of the receptor in the cytoplasm by a transducing mitogenic signal route. The involvement of the ras p21 protein in signal transduction is certain;
4. the proteins encoded by the mas and raf oncogenes are located in the cytoplasm. These are protein kinases that phosphorylate the proteins from serine and threonine residues, but their role in cell transformation is not known;
5. the proteins encoded by the myc, myb, fas, jun, erb A genes, the EIA adenovirus, the HTLV-I px virus, the polyomavirus large T antigen (PyLT), the SV40 large T (LT) antigen and the reticuloblastoma genes are located in the nucleus. Their location in the nucleus suggests that they can be involved in the DNA replication or transcription or in the maintenance of the nuclear structure [166].

ANTIONCOGENES or suppressor genes cause an inhibition of cell proliferation, and their absence or recessive forms favor malignant transformation. Oncogenes have a dominant character and they produce malignant transformation by their activation, while antioncogenes induce the same effect, by either their absence or multiple forms that have a recessive character. The existence of antioncogenes has been proved and is unanimously accepted, but the functioning mode of antioncogenes as well as of the proteins encoded by them is not clearly understood. The role of antioncogenes manifests by the inhibition of cell proliferation and not by the direct regulation of oncogenes [84].

Mendelian heredity of the predisposition to cancer can be divided in two groups:

• – the first group is formed by recessively inherited disorders, being constitutional in familial forms or somatic in sporadic cases, and causes the loss or alteration of a gene. The carriers of these inherited disorders exhibit DNA regulation defects, which leads to a higher incidence than normal of some cancers;
• – the second group includes predominantly hereditary disorders and is composed of oncosuppressor genes, being always somatic. The mechanism has a recessive character, so that in the case of oncogenes, the persistence of a normal gene is not sufficient to maintain the normal phenotype when one of the replicas is activated, while for antioncogenes, the conservation of one replica is sufficient. The malignant phenotype in these cases does only appear in the absence of the normal gene [101,102].

The detection of oncosuppressor genes has been possible by the combination of epidemiological familial studies with cytogenetics and molecular genetics. By using this complex methodology, the gene predisposing to retinoblastoma has been identified. Moreover, in the case of retinoblastoma and nephroblastoma, at the rupture or deletion sites, no active oncogene has been identified, which argues for the existence of antioncogenes. Likewise, in the case of experiments of hybridization between tumor and normal cells, the malignant phenotype regresses in the presence of normal cell genes. Another argument supporting the existence of oncosuppressor genes is the constant presence of chromosomal deletions in the majority of solid tumors, which suggests that some unidentified antioncogenes might exist [157,166, 171].

The implication of oncosuppressor genes in the regulation of normal growth, as well as of differentiations, has been proved by indirect and direct arguments.

The suppression of the transformed phenotype of hybrid cells, by the fusion of malignant cells with diploid fibroblasts from the same species, determines the appearance of hybrid cells that are non-malignant, as long as they retain certain specific chromosomes, donated by the normal parent [86]. When these particular chromosomes are lost from hybrids, due to the instability of chromosomes, the malignant phenotype reappears, and the segregating cell is able again to generate tumors in vivo.

The suppression of cancerous cell growth and the phenotype of transformation by contact with normal cells: when fibroblast cells transformed by the polyomavirus are cultured together with normal cells, the growth of the transformed cells will be inhibited.

Tumor inhibiting factors: certain proteins cause the suppression of tumor cell growth and inhibit cell fixation independence, in experiments in vitro. Injected in vivo, these proteins reduce the tumorigenicity, invasion and metastasis of cancerous cells [166].

The ß growth transforming factor, interferons, interleukin-2 and the tumor necrosis factor manifest inhibitory properties towards these tumors.

Different human tumor cells produce inhibiting tumor cell factors. On the one hand, these factors inhibit the growth of a variety of human tumor cells, but, on the other hand, they stimulate the growth of normal cells. Inhibiting tumor cell factors also inhibit the fixation independence, a property correlated with tumorigenicity.

Some properties of the products of protooncogenes are known, but very little is known about the functions of these proteins in normal cells and even less about the way in which the activation of protooncogenes and oncosuppressor genes in eukaryotes suggests that these genes serve important functions [166]. The cited authors mention the fact that the properties and cellular location of the gene products of oncosuppressor genes suggest several ways in which they can function.

Some oncoproteins are structurally similar to the secreted growth factors; moreover, oncogene activation can lead to the production of growth factors by the tumor cell itself. It has been suggested that autocrine mechanisms can play an important role in the control of tumor cell growth. Some oncoproteins are membrane receptors of the growth factor, for example, the oncoprotein is homologous to the epidermal growth factor receptor.

The ras, fps, abl, yes, fes oncoproteins are located on the internal side of the cytoplasmic membrane and are considered to be involved in the mediation of receptor signaling. Some oncoproteins are located in the cytoplasm (mos and raj), others are located in the nucleus (myc, myb, fas, jun, erb A, etc.). The role of these oncoproteins is not yet clearly understood.

For a certain time, it was considered that oncogenes act during certain specific stages of carcinogenesis, for example, myc would be an early gene, involved in the formation of “immortal” cells, and ras a late gene involved in tumorigenic transformation. It has been diagnosed that oncogenes cannot be classified based on these criteria, since there are oncogenes, such as ras or myc, that are involved in several phases or stages of cell transformation.

Oncoproteins act as oncogenes or oncosuppressor genes depending on the target cell.

The involvement of mutations in various phases of carcinogenesis is certain; it seems that some epigenetic events also contribute to the development of cancerous cells. These events cannot be avoided and include DNA, RNA and protein changes that can influence the cell phenotype, but not the genotype. Differentiation, which can be estimated to derive from a series of epigenetic changes, can modify the tumorigenic phenotype of the cells.

Tumor promoters and progressors are compounds that epigenetically affect carcinogenesis. Some of these, such as phorbol esthers, can cause non-typified proliferations of some cells and the differentiation of others.

The results of the investigations regarding oncogenes and oncosuppressor genes suggest possible applications in practical oncology. In this sense, the use for diagnosis (tumor classification, malignancy diagnosis, early metastasizing); prognosis (oncogene amplifications); therapy (monoclonal antibodies and radiochemotherapy); and epidemiology (genetic consultation) is intended. The determination of changes in oncogenes and oncosuppressor genes in tumors can be useful for diagnosis, prognosis and the therapeutic approach. The molecular hybridization technique involving tumor DNA or RNA, as well as immunohistochemical techniques, can be used in determining quantitative and/or qualitative changes of oncogenes and oncosuppressor genes, as well as their expression. Tumors can be located using monoclonal antibodies to which radionuclides are bound.

The discovery of cellular microRNA and its functions in particular has provided important data on cell biology, especially on the understanding of cancer and viral diseases. The let-7 microRNA has been demonstrated to be less abundant in patients with highly aggressive pulmonary cancer, compared to healthy subjects. In cancer cell cultures, microRNA has been shown to delay cell division, consequently being a tumor suppressor. Another important function of microRNA is to regulate the amount of messenger RNA produced in a cell. The actions and functions of microRNAs are multiple, on the one hand due to their diversity, and on the other hand due to the diversity of their targets, which makes them essential in cell differentiation. It has been demonstrated that each tumor type possesses a specific “expression profile” of microRNA, which opens the perspective for a certainty diagnosis of the tumor type and implicitly, for the application of adequate therapy. In addition, regarding viral cancers, we should mention that the pathogenic capacity of viruses might be related to their capacity to affect cellular microRNAs.

The change of oncogenes and oncosuppressor genes can be used for prognosis. For example, the overexpression of the ras gene in mammary cancer or of the myc gene in cervical or mammary cancer is characterized, based on findings, by unfavorable prognosis.

In therapy, the use of the DNA recombinant technology in order to produce proteins, which control cell growth, can be an efficient means of fighting malignant cells. Such tumor inhibiting substances include proteins, such as interferon, the tumor necrosis factor and interleukin 2. Potential therapeutic agents can be: tumor inhibiting factors (TIF-1; TIF-2, oncostatin M) and the tumor necrosis factor that reversibly inhibits the growth of cancerous cells, but not of normal adjacent tissue cells.

The results of in vitro experiments suggest that the reduced expression of oncogenes in a tumor can have a therapeutic value. The antibody cannot cause itself the complete elimination of the tumor; in contrast, a combination of antibodies in association with different epitopes have determined the eradication of the tumor in experimental animals.

The determination of the detailed structure of oncogenic products might allow the creation of adequate inhibiting substances, such as peptides or monoclonal antibodies, in order to inhibit their action or to associate them with a specific toxin or radionucleotide for the destruction of the cell [166].

MAIN EVENTS AT GENOMIC LEVEL DURING CARCINOGENESIS

2.3.2. Chronobiology of cancer

Chronobiology demonstrates that the adaptation of the organism to periodical environmental variations is related to the temporal genetic component.

HIPPOCRATES observed the existence of temporal relations in the clinical manifestation of certain diseases; thus, asthma episodes are more frequent and more severe at night, myocardial infarction occurs especially late in the evening or in the morning, gastric ulcer aggravates in spring and autumn, etc.

Bioperiodic activities are rhythmic: daily or circadian periodicity; cycles of second fractions up to several hours or ultradian cycles; rhythms that last for several days or infradian rhythms; approximately monthly or circamensual cycles; yearly or circannual, seasonal periodicity, etc.

Each organism is controlled by several biological clocks called oscillators, which are interconnected and hierarchized. Biological clocks are regulated through synchronizations that regulate the periodical variations of the environment (night fall, dawn rise, socioeconomic life requirements, etc.).

The chronobiology of cancer is demonstrated by its appearance and development in time, along successive stages: initiation, promotion, progression and malignant transformation. Chronobiology regarding the mechanisms of cancer genesis has been developed in theory, and at therapeutic level, chronotherapy has been developed.

The preferential proliferation of precancerous cells offers a higher probability of appearance of nuclear lesions, which will activate oncogenes or inactivate antioncogenes and, as a result, the mechanisms of malignant tranformation will be triggered. Epidemiological researches have demonstrated the higher incidence of hepatic cancer against the background of pathological conditions (cirrhosis, viral or toxic hepatitis), which involve chronic regenerative proliferations. In a desynchronized liver, cells have a higher probability to accumulate chromosomal aberrations in an increasingly high number.

Each cancer type shows circadian cell division variations. Hence, a drug could have a higher efficiency if administered at a certain time, and another drug at a different time. Researches have brought to attention the chronooptimization of treatments, which concerns the tolerance of the patient to the administered drugs, chronotolerance, as well as drug efficiency, chronoefficiency. A two-fold higher regression of neoplasms was experimentally found in animals treated according to the principles of chronotherapy, compared to animals treated classically. Similar results were obtained in patients with neoplasms, the survival of the subjects being twice as long compared to those receiving the classic treatment.

The difficulty of the study and of the determination of chronoefficiency consists in the great heterogeneity of rhythms that manifests in tumors. Hence, the interest in the development of adequate technologies involves, for each patient, the determination of the rhythmicity that orchestrates cell proliferation specific for each type of cancer. The optimization of tolerance can lead to an intensification of chemotherapy and, in the long term, to the optimization of efficiency. The use of chronoprogrammable groups of patients is intended for the continuous perfusion of anticancer drugs according to complex protocols, applicable on an outpatient basis. Chemotherapeutic doses may be significantly increased, without an increase in side effects, and even with their significant diminution.

By applying treatments according to the principles of chronoprogramming and chronotolerance in patients with neoplasms and metastases, a five fold lower toxicity was found, with the possibility of a significant increase of doses, and survival was significantly higher. At the same time, the surgical resection of tumor remnants left after drug treatment was possible.

In cancers that are not too advanced, when the disease maintains a good clinical index, tumor rhythms are close to those of the unaffected tissues of the patient. In subjects with advanced neoplasms and a poor clinical condition, rhythmicity is totally disturbed. Moreover, cell division rhythms at tumor level vary from one patient to the other.

Consequently, the individualization of treatments is required, and even the resynchronization of rhythms by drug therapy. However, there is a possibility that drugs might change the biorhythms of patients and consequently, the most tolerant hours and the best efficiency might change.

Once the existence of drug chronoefficiency was demonstrated, practical technologies for the treatment of neoplasms were initiated.

MILLS (1967) conceived the release of anticancer substances under the action of ultraviolet rays, from microspherules injected in experimental animals with Ehrlich tumors. Ultrasounds were applied every day at 23:00, which is the optimal hour for chronotolerance to cisplatin, in nocturnal animals. Results were more than encouraging.

In humans, polymeric matrices can be guided by arterial route to the tumor site, using screen guided probes. This method allows the exposure of the tumor to a high amount of cytostatic drugs with a reduced undesired effect, due to the possibility of setting the optimal hour for the treatment application.

It has been experimentally proved that as treatment advances in the group of cancerous cells, an early selection occurs and resistant cells appear. By the method of microspherules, from which the drug is released by means of ultrasounds, an efficient concentration of anticancer substances is ensured, which leads to the inhibition of cell divisions and the arrest of formation of resistant cells.