35 Cancer and AIDS

Dr. M. N. Gupta

1. Objectives

•  To learn the difference between benign and malignant tumours

•  To learn about distinguishing feature of a malignant cell

•  To learn about carcinogens and role initiators and promoters in carcinogenesis

•  To understand how DNA and RNA oncogenic viruses cause malignancy

•  To learn about HIV

2. Concept Map

3. Description

In a normal situation, cells follow the principle of live and let live. Cancer is an umbrella term for many diseases in which cells in particular organ/tissue undergo uncontrolled proliferation. The common feature is that they spread and invade the space occupied by other cells.

This transformation of the cell followed by invasion is termed malignancy.

In developed countries, infectious diseases, cardiovascular diseases and cancer remain causes of death with fairly high frequency.

Under normal circumstances, proliferation of cell is a highly regulated process. When this regulation is lost, cell proliferation causes tumours which are called neoplasm (new growth).

Non-invasive tumours which do not spread are called benign. Some neoplasm spread to surrounding tissues and are called malignant tumours or cancer.

Tumours formed from epithelial cells are called carcinoma. Those forming from stromal cells or mesenchymal cells are called sarcoma.

Growth kinetics of neoplasms

Tumours are, to begin with, essentially localized swelling. The causes are inflammation and/or infection and cell proliferation. Neoplasms are caused by cell proliferation and are sometime referred to as solid tumours.

The growth kinetics of solid tumours is biphasic. In the first phase, nutrients from intercellular fluid feed the cells by diffusion. In the second phase, tumour induces proliferation of host blood cells. The first phase is possible as active transport systems are more efficient in malignant cells as compared to normal cells.

This mechanism cannot sustain tumour growth beyond 1mm diameter size. Beyond this size, there are mass transfer constraints for inner cells of the tumour to have access to the nutrient.

Towards the end of the phase, tumour cells release angiogenesis factors which lead to formation of new blood vessels which ultimately reach outer layer of the tumour cells. Inner core cells die.

Let us consider 100 cells in an organ. Generally, 99% will differentiate and stop dividing. The single stem cell will continue dividing. This step is repeated.

At each step, stem cell will divide into one differentiated daughter cell and one stem cell.

In malignant tumours if all progeny continue dividing, tumours spreads very rapidly. If only a certain % of progenies divide, cancer spreads relatively at a slower rate.

Metastasis is spread of the malignant cells, first to the nearby cells. The second stage of metastatis occurs via invasion of a vessel and cancer cells entering into a lymphatic or blood vascular system.

Some metastasis occurs though exclusively via the lymphatic route. Lymphatic vessels carry intercellular fluid throughout the body and transport both particulates and fluid to lymph nodes. Cancer cells lodge in these lymph nodes.

Breast cancer is often spread by lymphatic route. In the microscopic view, the metastasis can be seen as a central light area which has abnormal epithelial structure.

The metastasis can spread via blood stream via two routes. First one, as already mentioned is the direct invasion of blood vessels. Alternatively, cancer cells in the lymph node may reach lymphatic (such as thoracic duct) from where it enters large veins connected to the heart. Spread of metastasis by blood stream route is called haematogenous metastasis.

As an example of the lymph node route, the cancer cells in the colon spread to the intestinal wall and reach local lymph node via lymphatic route. From there, it can spread to another organ like liver, brain, lung or bone.

The process of cells becoming malignant or cancerous is called carcenogenesis. A normal cell becoming malignant is called neoplastic transformation. A cancer cell gives rise to only another cancer cell. Hence, a clone of cancer cells is formed. At clinically detectable stage, for all practical purposes it appears that the tumor is derived from a single cancer cell, that is, it is monoclonal.

The probability that a particular tumour is malignant increases with its size. The correlation is different quantitatively for different cancers. A 10 cm tumor in breast has only 80% probability. Corresponding, for lung cancer, that probability belongs to 6 cm tumors.

Hematogenous cancers spread often to essential organs and are often fatal. Lymphatic metastases relatively are less grave. Leukemias can be considered a subclass of sarcomas and are malignant cells growing in the blood. The larger proliferation of blood cells makes patient’s cell appear as milky. Derived from latin, the word leukemia means white blood.

Cancer cells can be distinguished from a normal cell by examining the tissue under a microscope.

Cancer cells: rapidly grow, high nucleus: cytoplasm ratio, prominent nucleoli, more mitosis.

Appearance of invading cells in the tissue is the most valuable indication.

Cultured cells have been the most valuable system for understanding biology of the cancerous cells. Fibroblast is the dominating cell type. A fibroblast secrets the proteins which are part of fibrous connective tissues of animals.

Cultured fibroblasts are not as differentiated as true fibroblast and with a chosen trigger can be differentiated into different types of cells. So, these are closer to mesodermal stem cells.

Epithelial cells are another type which has been cultured and represents cell type arising from ectodermal or endodermal embryonic cell layers.

Both above cell culture grow on glass or plastic dishes by adhering to the surface as they secrete laminin, fibronectin and collagen.

Cell cultures from blood, spleen, bone marrow are non adherent type and such cultures are kept in suspensions.

Human cells during culturing have three phases: a growth phase, constant growth rate (upon continual dilution) and death. In mice and other rodents, dilution does not help. However, as a rare phenomenon, a variant (actually a variant with abnormal no. of chromosomes) may appear and grows forever. This is how immortal cell lines are obtained.

Non adherent blood cells from animal can be easily immortalized. Human cells and chicken cells are rarely immortalized. Rodent cells are much easier to create an immortal cell line.

3T3 mouse cell lines, for example, have proved immensely useful in cancer research.

Malignant transformation

Figure 8: Scanning Electron micrograph of NORMAL 3T3 cell: These are aligned and closely packed in an orderly fashion Scanning Electron micrograph of TRANSFORMED 3T3 cell: Virally transformed 3T3 cells are rounded, covered in hair-like and bulbous

Treatment of adherent cells such as viral infection, chemicals and irradiation can endow these cells to with the capacity to form tumours. Such processes are collectively called malignant transformations Large number of changes in the properties of the cell have been observed upon transformation.

Normal cells stop growing after they reach a cell density. Transformed cells show increased saturation density. Their requirements for growth factors/hormones etc. is less than normal cells or they secrete their own growth factors.

Transformed cells do not have requirement for adherence and can form colonies in agar suspensions. Their shape and appearance unlike normal cells form unstructured masses with multilayers. Transformed cells also no longer have contact inhibition of movement, unlike normal cells, while moving they can cross each other. Normal cells sort of recoil from each other.

Definite changes in the composition of cell surfaces have been observed upon transformation. Both sialic acid and ganglioside content decrease. Less lectin concentration are required for cell agglutination presumably as a result of increased mobility of glycoprotein on the cell surface.

More permeases (glucose transporter) are present on cell surface presumably because of the need in view of increased glycolysis. Actin microfilaments are absent.

Transformed cells secrets plasminogen activator which converts plasminogen into plasmin which helps penetration of basal lamina by the cells.

Altered gene expression is observed after malignant transformation.

About 1016 cell divisions take place in humans during their life time. Spontaneous mutation rate is estimated to be about 10-6 mutations per gene per cell division. This arises out of inherent inaccuracy in DNA replication.

The reason for a normal cell to become cancerous is not a single mutation. More frequent mutations do seem to be a contributing cause.

One evidence for this is that incidence of cancer rises steeply as a function of age. The correlation is more steep for carcinomas as compared to leukemia.

It is estimated that about 3-5 independent and random mutations have to occur in the same cell for it to develop malignancy.

The crucial question is what causes cancer? We have seen age as a factor. So is genetics.

More specifically, both environmental factors and some viruses have been identified.

The tragic part is that exposure to carcinogens is avoidable. Unfortunately that knowledge does not deter people. The correlation between lung cancer and smoking is well established. Yet people actually pay for buying cigrattes and other tobacco products!

Before the cancer can be clinically detected (and methods to detect it early are improving!), series of changes in the cells occur. Each step increases malignancy.

It should be understood that tumour (neoplasm) merely represent a cell mass growing rapidly. Malignancy is its invasion of tissue/organs. This generally means, some cells from the tumour coming apart and entering blood vessels or lymphatic vessels.

Epidemiological studies showed that some chemicals act as carcinogens. In fact, chemical carcinogenesis requires carcinogens and promoters.

It appears that carcinogens cause mutations whereas promoters triggers abnormal cell division.

Neither carcinogen alone nor in combination with promoters cause neoplastic transformation in cultured cell lines in vitro. The normal metabolism ironically transforms carcinogens into metabolites which interact with DNA. So, real or ultimate carcinogens are created by metabolism in vivo.

Apart from immune system detoxification mechanism protect animals when some chemicals are accidently consumed. These small molecular weight components, quite frequently are insoluble in water rich milieu. Detoxification mechanism involve conjugation with polar compounds, glycosylation and hydroxylation reactions so that the compounds are excreted out.

These detoxification reactions often involve reactive intermediate. If the step of their production is higher than their conversion to final excretion products, these accumulate to reach a level where they cause damage by mutating DNA.

Liver is the organ where many detoxification reactions take place. Aflatoxins are toxin from molds growing on grains and peanuts stored under humid conditions. In tropics, this is a serious contributing factor to incidences of liver cancer.

The insight into roles of tumour initiators (carcinogens) and tumour promoters has come from skin cancer elicited in mice. Even a single exposure to carcinogen in all likelyhood causes latent damage. Exposure to tumour initiator followed by tumour promoters leads to neoplastic transformation.

The exposure to promoter also has to cross a threshold limit. Promoters themselves are not mutagenic. Malignancy can also occur without promoter if exposure to the carcinogen occurs repeatedly at a significant level.

Promoters thus, sort of uncover, the damage done by carcinogens and cause cancers at a high frequency. For that, however, prior exposure to carcinogens is necessary.

Tumour progression refers to the phenomenon in which a single first aberrant cell develops gradually into a detectable cancer. External reasons like a casual exposure normally cause cancers after a long period of time. Incidences of leukemia in Hiroshima and Nagasaki peaked only after 8 years of atomic bomb dropped on the cities. Similarly, exposure to a carcinogen for a limited period of time to the industrial workers often takes 10-20 years before cancer is developed.

An illustration is the development of uterine cervix cancer. In the first stage of dysplasia, most superficial cells show signs of incomplete differentiation. In carcinoma cells in all layers proliferate but are undifferentiated. Malignancy onset is seen when cells cross the basal lamina to invade connecting tissues. Whole process may take place over several years.

Even at carcinoma in situ stage, surgical removal or destruction of the affected tissue is possible.

Interesting enough, at carcinoma in situ stage, even without medical intervention, it may not progress to the invasive stage or may even regress. However, it is estimated that in 20-30% of such cases, it will develop into full blown cancer without the medical intervention. At the invasive stage (malignant), surgical or other medical interventions are quite unsuccessful.

It is with that background that we can understand the role of tumour promoters, promoters stimulate cell division for cells which otherwise will be terminally differentiated.

Promoter cause formation of small benign tumours called papillomas. The number of papillomas formed directly depends upon the prior exposure dose of the initiator carcinogen. Many papillomas are clones of mutant cell formed as a result of exposure to the initiator.

Promoter encourage expression of genes in the mutant cells which influences cell proliferation. More mutations in the proliferating cells makes some cells malignant.

Epidemiological studies have revealed geographical regions of high incidences and low incidences for various cancers. Lung cancer has high incidences in new Orleans, USA among blacks and has low incidences in Chennai, India. Melanoma has high incidence in Queensland, Australia and low in Osaka, Japan.

Life styles seem to affect % incidences of at least some types of cancer. Mormons in Utah. USA are known for abstinance from many things. They have about 50% of cancer incidences as compared to USA in general.

Ames test proved very valuable in establishing carcinogenicity of many chemicals. It employs a Salmonella strain that has a mutant gene for histidine biosynthesis and hence requires histidine in the medium for its growth. Rat liver cell extracts is used to stimulate in the in vivo reaction which convert a potential carcinogen to the real carcinogen, the reactive intermediate. The potential carcinogen is added. By mutation, it can reverse the mutant gene responsible for histidine biosynthesis.

Ames test actually estimates mutagenicity of chemical. However, all initiator carcinogens are mutagenic. The results on mutagenicity with this bacterial system correlate well with mutagenicity of mammalian cells.

Rous was awarded the noble prize for his discovery of Rous sarcoma virus or avian Sarcoma virus (ASV) about half a century earlier. ASV is one of the RNA tumour viruses or oncogenic RNA viruses. As these contain reverse transcriptase, these are also known as Retroviruses.

Some DNA viruses like simian virus 40 (SV40) and polyoma virus can also cause malignancy and are called oncogenic DNA viruses.

Infections by these oncogenic viruses cause permanent transformation of the animal cells which become cancerous.

Oncogenic DNA viruses (Papova viruses) infection leads to integration of their DNA with host DNA at random sites.

Oncogenic DNA viruses code for both cell surface and nuclear antigens.

In oncogeneric RNA viruses (Retroviruses), the retroviral DNA made by reverse transcriptase is transcribed only after integration with animal cell genomes retroviruses do not seem to be a major cause for common cancer in humans. However, their studies with other animals have provided valuable molecular level insight into cancer.

The cellular antigens expressed at specific stages of differentiation are called differentiation antigens. These can also be found on malignant cells and can be identified by use of monoclonals.

Use of monoclonals also allows insight into the stage in differentiation at which cell become malignant. For example, thus approach has shown that majority of T-cell leukemias are caused by transformation of prothymocytes or early thymocytes.

Papoviruses encode transforming proteins

SV40 encodes two early proteins called T (large T) and (small) t. polyoma virus makes three early proteins called T. mid-T and t.

The T antigens are 90,000 kDa nuclear proteins and play key role in transcription of viral DNA and their replication. The mid-T of polyoma is 45kDa membrane bound protein. In SV40, some T antigen is bound to membrane. The t protein are cytoplasmic and are of about 20 kDa.

All 3 kinds of proteins have independent roles in causing malignancy. In case of polyoma virus in which genes have been supplied out, the mid T-proteins can transform rat 3T3 cell lines but transformed cells will still require all growth factors. T-protein confer the property of growth in low serum but such cells are not morphologically transformed.

Table 7: Proteins encoded by SV40

In SV40, different parts of T-protein play different role in transformation.

Normal vertebrate genome contains potentially cancer causing genes.

RSV was found to contain src which is not found in non transforming retroviruses. Src was also found in host chicken DNA and in fact in DNA of all vertebrates and some invertebrates.

Oncogens are cancer inducing genes. (Gk. Onkos means bulk or mass!). src of the RSV is called v-src to distinguish from the animal one which is called c-src.

Retroviral oncogens can be classified into five classes. In all cases, they have a counterpart in normal animal cell DNA.

Many code for the tyrosine kinases and thus are involved in signal transduction. In other cases, their products are similar to growth factors expressed by normal growth animal cells. Other express protein similar to growth factor receptors Transformation by retroviruses results in both qualitative to quantitative changes in gene expression. Oncogens often code for proteins involved in growth and development.

Acquired immune deficiency (AIDS)

It was in 1981, that first cases of a new disease were reported. This disease has come to be known as acquired immune deficiency syndrome The reason we are discussing this in this module is because it is now known that AIDS is caused by a retrovirus. This discovery was made by Luc Montagnier and Robert Gallo.

It is worth noting that in old days, identification of the cause of a new disease often used to take decades. In this case availability of current tools resulted in the identification of AIDS virus within 2 years of the first few reported cases of the disease.

HIV (human immunodeficiency virus), the cause of AIDS is similar to human T-cell lymphotropic virus I (HTLV-I). Thus, HIV also infects T-cells.

It is estimated that about 40 million people are infected with HIV and about 2.5 million cases are added each year by AIDS. Just to provide a perspective, about 2.2 million people die each year of diarrhea in the developing world.

HIV has an envelope of bilayer membrane containing integral glycoprotein gp41 which is linked by S-S bond to gp120.

HIV gp120 interacts with CD4 on T-cell surface, membranes fuse and viral core enters cytoplasm of the host T-cells.

The 9.7 kb RNA core of HIV is more complex than other retroviruses. The unusual genes are sor, trs, tat and 3’orf.

•  tat stimulates viral mRNA synthesis

•  trs increase the expression of gag, pol, env and sor genes.

•  env has very high mutation rate.

•  gp 120 show high degree of variability in amino acid sequence when isolated from the same patient at different times.

No successful vaccine for AIDS is available at present although several drug cocktails have shown some encouraging results in some cases.

Immune system does try to destroy malignant cells. Both NK cells and adaptive immune system components try to control cancer. Mechanism by which tumour cell escapes destruction may involve induction of tolerance, lack of expression of MHC class I protein, selection of tumour cells with antigens which fail to elicit strong immune response, immuno suppression and expression of Fas T ligands which causes T-cell apoptosis by interacting with their Fas surface receptors.

Summary

•  Benign tumours and malignant tumours

•  Distinguishing properties of a malignant cell

•  Chemicals as carcinogens

•  Role of DNA and RNA viruses in cancer

•  HIV