Cellular Biology

1. Cellular Biology School of Life Sciences Shaanxi Normal University 1

2. CHAPTER 12 TUMOR CELLS AND GENE THERAPY OF TUMORS

3. Tumor (neoplasm) is the disease involved gene mutation, but it can not be inherited. Tumor is the abnormal proliferation and differentiation of cells when the tumor inducer factors induced genes mutated and lost the normal regulation to cell growth. Grow slowly Benign tumors Obvious border with normal tissue Tumors No metastasis Grow fast Malignant tumors (cancers) No border with normal tissue Metastasis Cancer (carcinoma) is the one of major killers to people. It is second people killer in US (First one is cardiovascular diseases) and first in China. Almost every organ or tissue can be developed as cancer, but most popular cancer easy developed organs include lungs, stomach, colon-rectal, liver, breasts, blood cells, and others.

4. Parenchymal part: Tumor cells. Tissue specificity. Tumor Is composed of connective tissues, vessels, Interstitial Part: and lymphatic vessels. Support tumor with nutrition. No tissue specificity.

5. I. The major features of cancer cells Generally, cancer cells keep three basic characters as following: 1.unlimited life; 2. metastasis; 3. no contact inhibition. The characters of morphology: 1. Usually, cancer cell is larger than the normal cells of same tissue with size heterogeneity. The ratio of nucleus/plasma is significantly raised to 1:1. 2. Morphology of nuclei are heterogeneity with macronuclei, binuclei, and multinuclei. The chromosomes are aneuploidy with some absence or addition. If the described as above happened in normal cell, the cell will turn to apoptosis. But in cancer cells, no apoptosis happened because of apoptotic signals inhibited. 3. Mitochondria present polymorphosis, swelled, regenerated. For example, macromitochondria in liver cancer cells. 4. Cell skeleton is changed and the tumor associated antigen is appeared. The characters of physiology: 1. Cell cycle gets out of control. 2. Metastasis. Cell adhesion and linkage associated molecules, such as ECM and CAM, are mutated or absented, and relative signal pathways are blocked, so, cancer cells are easy to move off from tumor tissue. Most of cancer cells secret some enzymes to enhance migration and metastasis.

6. 3. Loss of contact inhibition. 4. Loss of anchorage dependence. Anchorage dependence means that cells must attach to some special ECM to grow and inhibit apoptosis. Tumor cell can grow up on agar, methyl cellulose, and others. 5. Dedifferentiation. More than 20 embryonic isozymic proteins are expressed in cancer cells. For example, α- fetoprotein (AFP) can be detected in the blood of liver cancer patients. 6. The dependence on GF is decreased. The selfcrine way and proliferation signal way are not depended on GF. Cancer tissue can secrets VEGF (Vascular endothelial growth factor) to enhance tumor growth. 7. Active metabolism. The activity of DNA and RNA polymerases in cancer cells is higher than in normal cells, and the nucleotide degeneration is inhibited. 8. The synthesis of protein is significantly enhanced and the degeneration of protein is inhibited in cancer cells. This feature results in cachexia for patients. 9. Disorders of mitochondrion functions. Tumor energy supply is mainly depended on glycolysis. 10. Transplantability. Cancer cells can be transplanted into other individuals or animals without immunological rejection. So, cancer cells can be transplanted into mice to form the transplanted cancer models.

7. Metastasis

8. Loss of contact inhibition

9. Migration in cultured Caco-2

10. II. Oncogenesis Oncogenesis includes mutation, latency, carcinogenesis, and development. Initiation mutation will not form tumor without a suitable environment, this stage is called as latency. Carcinogenesis means that the mutated cell will start proliferation with carcinogenic factors, such as GF. Carcinogenesis is reversible if the carcinogenic factors are stopped. Development means that the tumor is becoming powerful to invade and metastasis. Development is irreversible. Oncogenesis is based on the mutation of many genes during a long time. The inner causes of oncogenesis The oncogenesis of malignant tumor is involved with the mutant accumulation of many genes including proto-oncogenes and cancer suppressor genes. Proto-oncogenes: Proto-oncogenes are the genes that are important to maintain the cell proliferation. But, mutated proto-oncogenes can cause oncogenesis.

11. The products of proto-oncogenes include ① GF; ② GF receptors, such as fms, erbB; ③ Protein kinases and other signal transduction molecules, such as Src, Ras, and Raf; ④ Cyclin, such as bcl-1; ⑤ Regulator of apoptosis, such as bcl-2; ⑥ Transcription factors, such as myc, fos, jun. Oncoviruses contain many proto-oncogenes in their genomes. Especially, reverse transcription viruses can recombine their proto-oncogene sequences into host genomes. Most of human proto-oncogenes were from oncoviruses probably. We call the proto-oncogenes that exist in normal cells as c-onc, in viruses as v-onc. The sequence of c-onc contains introns, and v-onc contains no intron. The detail about oncoviruses and proto-oncogenes will be presented in your Virology later.

12. The products of proto-oncogenes

13. Functions of proto-oncogenes

14. Cancer suppressor genes: Cancer suppressor gene is also called anti-cancer gene. They are contained in normal chromosomes probably. The loss or mutation of cancer suppressor genes can activate proto-oncogenes and cause oncogenesis. The products of cancer suppressor genes can inhibit tumor cell proliferation and metastasis, and promote differentiation. The products of cancer suppressor genes include ① Regulators of transcription, such as Rb, p53; ② Down regulator of transcription, such as WT; ③ Cyclin depend kinase inhibitor (CDKI), such as p15, p16, p21; ④ Inhibitors of signal pathways, such as NF-1, PTEN. ⑥ DNA repairing factors, such as BRCA1, and BRCA2; ⑥ The molecules of signal pathways associated with development and stem cell proliferation, such as APC and Axin. The ways of inactivation of cancer suppressor genes: ① If both anelle genes of cancer suppressor gene are mutated or inactivated, the cancer suppressor gene is inactivated; ② The dominant negative of cancer suppressor genes. The mutated copy of cancer suppressor gene can inactivate the wild type copy of cancer suppressor gene. This inactivation is called as dominant negative. ③ Haplo-insufficiency theory. The single copy of some cancer suppressor genes can not be expressed enough to maintain normal cell cycle. For example, the loss of one copy of DCC gene can cause cell contact inhibition inhibited to form a malignant cell clone.

15. Retinoblastoma gene (Rb gene) is the first cloned cancer suppressor gene. The mutation of Rb gene causes retinoblastoma. Sporadic Rb is happened in single eye usually. Genetic Rb is developed in dual eyes at about three years old. APC gene (adenomatous polyposis coli) was named with the tissue name where it was firstly identified. APC gene is located on chromosome 5q21-22. APC protein can bind to β-catenin to enhance β-catenin degeneration. The accumulated β-catenin can bind to T cell factor (TCF) to promote relative gene expressions. The peptide sequence of DCC gene (deleted in colorectal carcinoma) is very similar to nerve cell adhesion molecule(N-CAM). Inactivation of DCC causes the changes of cell adhesion, contract inhibition, and mobility, that pushes cell turn to malignant growth and metastasis. The loss rate of DCC in stomach cancer patients is about 40%~60%.

16. The mechanisms of sporadic and genetic function loss of Rb

17. The fuction of Rb gene Rb binds to E2F in G1 phase to inhibit the activity of E2F. Rb is phosphorated by CDK2 in G1/S phase and releases E2F (transcription factor) out to promote protein synthesis.

18. Activation of proto-oncogenes: The oncogenesis of malignant tumors is based on the activation of proto-oncogenes and the inactivation of cancer suppressor genes. Proto-oncogenes can be activated by variety of mutations on proto-oncogene or the gene’s regulator. 1.Site mutation: The mutation for ras family is site mutation. For example, The c-Ha-ras gene cloned from carcinoma of urinary bladder is different with the normal c-Ha-ras by one base pair only. 2.DNA rearrangement: The gene DNA of the normal proto-oncogenes can be rearranged by shift, translocation, or location reverse. The rearrangement can put proto-oncogene to the down stream of some powerful promoter, and causes excessive expression. The shift on the chromosome of Burkitt lymphoma moves c-myc close to the regulator of the heavy chain of immunoglobulin (Ab) that is very actively expressed always, and in addition, there is a powerful enhancer for this promoter. So, the shifted c-myc will be excessively expressed to cause oncogenesis.

19. 3. Inserted activation: Some reverse transcription viruses containing no v-onc can insert their provirus DNA into host DNA to cause inserted activation. MoSV (Mosaic virus) can insert its LTR sequence that contains powerful promoter and enhancer into a site closed to the upper stream of c-mos in fibroblast of mouse to cause a mouse sarcoma. The ALV (Avian leukemia virus) contains no v-onc, but it can insert to the upper stream of c-myc to cause c-myc expressed excessively. 4. Amplification of genes: The amplification of some proto-oncogenes is a popular event in some blood tumors. c-myc can be excessively amplified 8-32 folds in leukemia. The parts of chromosome are amplified include ①Double minute chromosomes (DMs) that are the micro chromosomes without centromere and distributed in pair. ②Homogenously stained region (HSR). HSR is formed by the amplification of chromosome region. ③Unequal sister chromatid exchange (USCE). USCE happens in G2 phase causing an allele chromosome in one generated cell is short and another allele chromosome in another generated cell is long. DMS and HSR are the popular types of amplification. The c-myc mRNA in the rectal carcinoma containing DMA or HSR is 30 folds higher than normal. 5.Low methylation of proto-oncogene: The cancer inducers can get the methylation of proto-oncogene decreased to cause oncogenesis.

20. The pathways of activation of proto-oncogene

21. The DMs (yellow) of amplified c-myc (from Molecular Biology of the Cell. 4th ed. 2002)

22. The HSR (yellow) of amplified c-myc (from Molecular Biology of the Cell. 4th ed. 2002)

23. The external causes of oncogenesis 80% of human tumor are caused by external tumor inducers. These external causes can be sorted as three types with their quality: chemical, biological, and physical inducers. By their functions, they can be sorted as initiator, enhancer, and complete carcinogen. Initiators means some molecules or conditions that can change the DNA sequence by one time of touch usually. Initiation is not reversible usually. Enhancer can not cause oncogenesis, but can push oncogenesis after initiation. Dimethytenzanthracene (DMBA) is a cancer initiator, and croton oil is an enhancer. Smear DMBA on to the skin of mouse, then smear croton oil onto same area of skin, the mouse skin cancer will be caused. Some enhancers are specific to enhance special cancers, for examples, saccharin enhances the oncogenesis of urinary bladder carcinoma, phenobarbital enhances the oncogenesis of hepatocarcinoma. Complete carcinogen means some molecules or bio-molecules with the strong initiating and enhancing function to cause oncogenesis. Complete carcinogens include polycyclic aromatic hydrocarbon, aromatic amine, amine nitrite, carcinogenic viruses, and others.

24. Initiator and enhancer (promoter) (from Molecular Biology of the Cell. 4th ed. 2002)

25. Chemical carcinogens: ①Amine nitrites: Strong carcinogens. Amine nitrites are contained in many quality changed vegetables and foods. Amine nitrites can cause variety of tumors of digestion system and kidneys. ②Polycyclic aromatic hydrocarbons:Polycyclic aromatic hydrocarbon can cause skin carcinoma and sarcoma. Polycyclic aromatic hydrocarbons are contained in the waste gas from car, smoke, pitch, smoked foods, and others. ③Aromatic amines: Aromatic amine can cause oncogenesis of urinary system. ④Alkylating agents: Alkylating agents can cause leukemia, cancers of lungs and breasts. ⑤Aminoazo-compounds: Aminoazo-compounds cause hepatocarcinoma. ⑥Base analogs: The DNA base pair can be replaced by base analogs, such as 5-FU and others, and interfere DNA replication. ⑦Chloroethylene: Polychloroethylene is the most popularly used plastic material composed of chloroethylenes. Chloroethylene is the carcinogen for the tumors of skin, lungs, and bones with a long time of latency. ⑧Heavy mental: Chromium (Cr), Nickel (Ni), Arsenic (As), and others.

26. Chemical carcinogens cause human tumors with complicated mechanism of serial steps of reactions. Some chemical carcinogens cause human tumors directly, we call them as direct carcinogen. Most of chemical carcinogens cause human tumors indirectly by a serial of transformation or modification to form the terminal carcinogen. We call these carcinogens as indirect carcinogen. P450 enzyme system is important to the oncogenesis induce by indirect carcinogen. Terminal carcinogens can interact with DNA, RNA, and proteins to cause base pair shift, absence, DNA conjugated, DNA broken, and abnormal chromosome. Chemical carcinogens can inhibit methylase to cause a low methylation, and activate proto-oncogenes. Direct or indirect carcinogens cause DNA mutated, and we call them as genotoxic carcinogen. The chemical carcinogens mentioned above are those. The carcinogens for the cancers of breasts, prostate, and uterus are the hormonic sterol compounds that do not cause DNA damaged, but enhance cell proliferation. We call them as non-genotoxic carcinogen.

27. Bio-carcinogens: Bio-carcinogens include viruses, bacteria, and fungi. Virus is most important bio-carcinogen. 1. Oncoviruses: ① Reverse transcription viruses: HTLV (human T lymphocyte leukemia virus), ATLV (adult T lymphocyte leukemia virus), and HIV. ② HBV. ③ HPV (human papilloma virus). ④ Epstein－Bars virus (EBV). Virus infection is very important to cause oncogenesis. There are many complicated contents will be presented in your Virology. 2. Fungi: Tens of fungi have been identified as bio-carcinogens so far. Aflatoxins are important carcinogen from the foods, especially peanuts and corns contaminated by fungi. Aflatoxin B1 is the one of the strongest hepatocarcinogen we know so far.

28. Some oncoviruses

29. Physical carcinogenic conditions: 1. Ionizing radiation: Ionizing radiation can cause the tumors of variety of organs or tissues. There are distinguished instances fro that: The death of Mrs. Curie, and the high incidence of leukemia in Japan after the nuclear bomb attack in Second World War. Ionizing radiation cause chromosome and DNA mutated, or activate proto-oncogenes. Ionizing radiation can cause the malignant tumors for breasts, lungs, skin, bone, leukemia and lymphoma. 2. Ultraviolet ray: Ultraviolet ray cause DNA broken and conjugated, chromosome mutated. Ultraviolet ray inhibit immunity to tumor immunity escaped. All above benefit oncogenesis.

30. The initiation and development of tumors There are two theories to understand tumor initiation that are from the dedifferentiated somatic cells, or from stem cells. Somatic cell is not of the ability of self renewal. Stem cells always keep self renewal ability, so, mutation is easy to be accumulated in stem cells. Many scientists believe that the tumor initiation is from stem cells. Some tissues, such as liver and kidneys, are not of ability of self renewal, but their cells can turn to division under some special condition, so, these cells are also the target cells for carcinogens. Stem cells have some similarities with tumor cells: 1. Self renewal and unlimited proliferation; 2. High telomerase activity; 3. Same signal ways to regulate self renewal, such as Wnt, Hedgehog, Notch, NF-κB.

31. Signal pathways to cause oncogenesis (From Molecular Biology of the Cell. 4th ed. 2002)

32. Most of tumors are initiated from monoclonal cell. Different isozymes are expressed in different cells. The isozymes are isolated from a tumor are same one. This result indicates that all cells of a tumor are proliferated from a monoclonal cell. Some tumor cells are of strong proliferation ability, we call them as tumor stem cell. The aim of tumor therapy is to kill tumor cells as much as possible. Many scientists believe that when a tumor became small means the therapy is effective. But, most of cases will relapse again. This recurrence means that tumor stem cells were not killed completely. Many scientists think that the tumor therapy should kill tumor stem cells as much as possible in spite of the tumor is not becoming small.

33. Female Barr body (Sex chromatin) indicates that tumor is initiated from monoclonal cell (From Molecular Biology of the Cell. 4th ed. 2002)

34. III. The diagnosis and therapy of tumor Like the diagnosis of other diseases, the medical history and sign checking are the basic methods to the diagnose tumors. The methods to diagnose tumor include X-ray photo, ultrasonic image, endoscopic image, histochemical section, biopsy, cellular biological items, CT, and serial special laboratory items including tumor specific marker. The diagnosis of tumor, especially malignant tumor, is very important to save patient’s life or delay his/her death from tumor. The diagnosis of tumor should be figured out as early as possible. As a non-medical student of life sciences, you should know how important is the tumor marker to the early diagnosis of tumor, and what the tumor markers are.

35. Tumor marker Tumor markers are the special proteins or polypeptides from tumor tissue. Good tumor marker should be isolated or detected out from specific tumor only! Tumor marker can be used to tumor diagnosis, therapy, therapy efficiency evaluation, and prognosis evaluation. So far, the following types of tumor marker have been researched: ① Oncofetal proteins; ② Tumor-associated antigens; ③ Enzyme; ④ Special serum proteins; ⑤ Hormones; ⑥ Proto-oncogenes, tumor suppressor genes, and their products. Tumor markers are used to diagnose tumor for many years. But, no any tumor marker is very specific, sensitive, or convenient to be used so far. So, several markers are used together to co-diagnose a tumor usually. To find more specific, sensitive and convenient tumor marker is still a very important and valuable research project now.

36. Oncofetal proteins During the individual development, some proteins are expressed in fetal stage. But, some tumor cells can be dedifferentiated to express these proteins. We call these tumor expressed fetal proteins as oncofetal proteins. Alpha-fetoprotein (AFP): Normal reference is <5.8μg/L in serum after one year post birth. High AFP expression is the specific sign to primary hepatocarcinoma. Carcinoembryonic antigen (CEA) is the glycoprotein located on the colon carcinoma and embryonic colon epithelial cells. Normal reference is <2.5μg/L in serum. Pancreatic oncofetal antigen (POA) is a glycoprotein with normal reference of <7U/ml in serum. POA is specific marker to pancreatic cancer.

37. Carcinomic antigen or cancer antigen (CA) Carcinomic antigen is the carbohydrate antigen (CA) that is content of cell membrane. CA was identified by monoclonal antibody. CA15-3: CA15-3 is secreted by the secreting epithelial cells of breasts, lungs, uterus, stomach and intestine. This antigen appears at high level (>30U/ml) with cancers of breasts, lungs, prostate, ovary, stomach and intestine. CA15-3 can be used as a good parameter to evaluate the prognosis of surgery on breasts cancer. CA19-9: CA19-9 is glycolipid with normal reference of <37U/ml. 85％-95％ of pancreatic cancers present high level of CA19-9. A high level of CA19-9 after the surgery operation on pancreatic cancer indicates the recurrence. CA125: CA125 is the marker for the cancer of ovary and uterus with normal reference of <35U/ml. CA50: CA50 is the marker for pancreatic cancer and colorectal carcinoma with normal reference of <20U/ml. PSA (prostate specific antigen): PSA is the marker for prostate cancer with normal reference of <2.5μg/L.

38. Enzymes as markers: The activity of some enzymes or the spectrum of some isoenzymes can be changed in tumors. PAP (prostatic acid phosphatase): Eosin phosphatase is the marker of lysosome. PAP is 100-1000 folds higher in prostate than in other tissues. PAP level in original prostate cancer can be normal, but tens folds higher in metastasis of prostate cancer than normal. LDH (lactate dehydrogenase): LDH activity can be increased in cancers. But it is high also in some other diseases, such as cardiovascular diseases. The activity of LDH4 and LDH5 can be higher than normal in malignant tumors with lower activity of LDH1 and LDH2. The activity of LDH5 is higher than LDH4 in primary hepatocarcinoma, but the activity of LDH4 is higher than LDH5 in secondary hepatocarcinoma.

39. AFU (α-L-fucosidase): AFU is the marker for primary hepatocarcinoma. ALP (Alkaline phosphatase): ALP is the marker for primary bone cancer and the metastasis of liver and bone from the cancers of other tissues. γ-GT (γ-glutamyl transpeptidase): γ-GT is the glycoprotein on cell membrane. 4％-30％gradient PAGE can sort serum γ-GT as 12-13 isoenzyme bands. γ-GT I', II and II' is the primary hepatocarcinoma. NSE (neuron-specific enolase): NSE is the marker for neuroblastoma and microcytic pulmonary carcinoma. GST (glutathione S-transferase): Isozymes of GST include GSTα, μ and π. GST-π can be used as the marker of malignant tumors of digestion system. Telomerase: Telomerase is a reverse transcriptase to repair telomere sequence. Telomerase is almost inactivated in normal tissues, but activated in tumors.

40. Hormones: The harmonic factors in the cancers of non-endocrine tissues are called as ectopic hormones. The increased hormones in endocrine cancers are called as abnormal hormones in situ. Both of them can be used as the markers for cancers. CT (calcitonin): The serum CT is obviously increased in medullary carcinoma of thyroid gland, lung adenocarcinoma, and microcytic pulmonary carcinoma. Serum CT can be high also in other tumors and diseases. hCG (human chorionic gonadotropin): hCG can be increased in pregnancy, chorionic epithelioma, and other cancers of genital system. Other hormones: human placental lactogen (HPL), adrenocorticotropic hormone (ACTH), growth hormone (GH), parathyroid hormone (PTH), and others.

41. Plasma proteins: Protein markers were used as tumor marker earlier than others. The specificity of protein markers is not so good, but they are easy to detect. β2m (β2-microglobulin): β2m is expressed on the surface of most of nucleated cells, and the light chain of HLA (human lymphocyte antigen). β2m is used to co-diagnose the malignant tumors from lymphocytic system. Fer (ferritin): Fer is used to co-diagnose the hepatocarcinoma, breasts cancer and other cancers. BJP (Bence-Jones protein): BJP is the specific marker for multiple myeloma.

42. Therapy of tumors Therapy of tumors is aimed to clean all tumor cells or decrease the tumor cells as much as possible to maintain the life of patients in his/her whole life, or change the quality of tumor cells to save the life of patients. The routine treatments for tumor therapy include surgery, radiation therapy, and chemical therapy. The successful tumor surgery therapy is depended on early diagnosis, individual features and the features of tumors. Radiation therapy (X-ray or γ-ray) takes severe side effects. Chemical therapy means that some chemical medicines (5-FU, Oxaliplatin, and others) are used to inhibit or clean the tumor cells. But, usually, chemical therapy takes so severe side effects that cause patients died from the chemical therapy, not from cancer.

43. Generally speaking, by our current medical technology level, no any good, effective or perfect method to save the life of malignant tumor patients! Tumor has been, and will be a great research target to us for a long time! Now, some new therapy methods are researched and developed. For examples, inhibition of vascular genesis, enhancement of tumor differentiation, immunological treatment, and gene therapy. Every new therapy method is the current hot research project, especially gene therapy. I introduce the gene therapy research to you briefly as the follows: Target therapy: Target therapy means that delivery the effective medicine, bio-product or other unspecific agents to a tumor target directorially by some special methods. For example, by the gene engineering (gene modification, recombination, or mimicry), the one of arms (Fv, ScFv) of IgG can be changed to bind target cell, another one can be changed to bind a agent molecule. This complex can shoot the target like a missile.

44. A bio-missile

45. Gene knockout: Gene knockout means that some harmful or benefitial gene is artificially damaged or blocked completely and persistantly. It can be used to construct cell or animal models for reasearches. Inserts

46. Antisense DNA: Antisense DNA is designed to inhibit or interfere some gene’s expression using a oligonucleotide matched to the target sequence of the gene’s mRNA or genome DNA (16bp). RNAi (siRNA) or Antisense RNA: Genomic DNA mRNA Antisense RNA Synthesized Expressed RNAi or siRNA

47. Antisense DNA: Genomic DNA mRNA Synthesized Antisense DNA

48. The presentation about Cellular Biology is ended here. We hope you, every one, will pass the testof examinationperfectly!