Tumor Cell Growth
and Cell Kinetics
Julena Lind
N UNDERSTANDING of cancer requires a basic knowledge of the differences between normal and malignant cells. Understandingnormal cell structure, growth, and kinetics through the life cycle is the critical first step in that knowledge. This article presents the characteristics of malignant cells, in vivo and in vitro, tumor growth and kinetics, and finally a summary of the biology underlying cancer treatment.
A
IN WV0 CHARACTERISTICS
OF CANCER CELLS
Most of the understandingof cancer cell growth has been achieved by changing normal cells into malignant cells outside of the human body. In order to apply this research to human beings, we must know how the behavior of cells in vitro applies to the behavior of cells in vivo. Increased Cell Renewal And Unresponsiveness to Growth Controls
Cell growth is a carefully regulated processthat respondsto specific needsof the body. When the controls that regulate cell replication break down, a cell begins to grow and divide without regard to the needfor more cells of its type. The descendants of such a cell inherit the propensity to replicate, and the result may be a clone of cells able to expand indefinitely. Most human tumors arise in renewing cell populations or in tissue that has been exposedto a chronic irritation that causesproliferation. Stem Cells
In normal tissues, new cells are derived from a small number of stem cells that have a high capacity for cell proliferation, but their actual rate of cell division is usually quite low unless there is tissue injury or demand. Stem cells have two critical functions: to generate a large family of descendants that will perform the function of the tissue and to renew themselvesso that a stable number of stem cells remain (Fig 1). i Monoclonality
and Tumor Heterogeneity
Tumors are thought to be monoclonal: that is, they arise from the transformation of a single precursor cell, which then proliferates to form a neoplastic clone. Transformation is most often thought Seminars in Oncology Nursing, Vol8, No 1 (February), 1992: pp 3-g
to be a result of carcinogenic events occurring in a single stem cell.2 However, when individual cells within tumors are studied, it is found that the tumor is comprisedof subpopulationsof biologically diverse cells that have differentiated from the same stem cell clone. The biological variability of cells within a tumor is referred to as tumor heterogeneity.374 Invasiveness
Most tumors pose little risk to humans because they are localized. Benign tumors contain cells that very closely resemble normal cells and, in fact, function like normal cells. Ordinarily, these benign tumors, like normal cells in tissues, remain localized in the appropriatetissues. However, malignant tumors are characterizedby their invasiveness and ability to spread throughout the body. They contain cells that are usually less well differentiated than normal cells, and their biological properties change over time. Changes in Cell-to-Cell Interaction
Invasiveness and metastasis are the result of changes in cell-to-cell interactions. Normal cells are restrictedto certain organsor tissuesbecauseof cell-to-cell recognition or by the presenceof physical barriers such as the extracellular matrix. Cells recognizeeachother by processesthat take place on the surface of the cell. These processes dictate which cells bind together and interact.’ Normal cells recognize and respectthe boundaries and the territory of the cell surrounding them. In cancer cells, surfaceenzymesare altered and recognition is impaired resulting in a number of biological changes including loss of contact inhibition. ’ The response to physical barriers is also changed;for example, many metastatictumor cells From the Department of Nursing, University of Southern California, Los Angeles, CA. Julena Lind, MS, RN: Interim Chair, Department of Nursing, University of Southern California. Address reprint requests to Julena Lind, MS, RN, 320 West 15th St, Los Angeles, CA 90015. Copyright 0 1992 by W.B. Saunders Company 0749-2081192/0801-0002$5.OOlO
3
JULENA
Molurolion without division
Functional finite
cells
with
lifetime
have the ability to “digest” their way through extracellular matrices by secretion of proteolytic enzymes or proteases.6 Escape From Immune Surveillance
Another recognized property of malignant cells in vivo is their ability to elude the immune system. It might be expected that the alterations in cell structure associatedwith malignancy would lead the immune system to recognize tumor cells as foreign. The blood even contains a “natural killer” cell that can recognize and kill many types of tumor cells while sparing normal cells. Despite this, tumor cells find ways to elude immune detection.’ Appelbaum describes this phenomenon elsewherein this issue. ONCOGENES
It is now clear that genetic changesare responsible for neoplastic transformation. Research in molecular biology has been focused largely on identifying the genesthat play a causal role in tumor development. Genesinvolved in the induction of neoplastic transformation were first identified in tumor viruses and later in human tumors. Oncogenesare genesthat promote growth; their protein products “turn on” cell division. When normal growth genes mutate to form oncogenes, their abnormal protein products may lead to tumor growth.’
LIND
Fig 1. The hierarchy of cells in renewal tissue. The mowlation is maintained by clonal xpansion from a small number of stem cells. (Reprinted with permission.‘)
Most oncogenesare derived from normal cellular genescalled proto-oncogenes.Proto-oncogenes are not cancer-causingin their normal cellular environment, and most of them presumably fulfill essentialphysiological functions in cell growth and development. The transition from normal genetic material, a proto-oncogene, to an oncogene includes changesin gene control and gene expression, but it is not yet clear exactly which changes in structure and expression are necessary.Not all human tumors contain identifiable oncogenes.9 Oncogenes and Tumor Growth
Some experiments have linked growth factors and oncogenesand suggesta relationship between malignancy and the loss of mechanismsthat control cell proliferation in normal tissues.lo-‘* Although growth control is not completely understood, there are at least three types of proteins that participate in the process:growth factors, growth factor receptors found on the plasma membrane, and intracellular proteins. Oncogenesand growth control factors are discussedin detail by Yarbro elsewherein this issue. CHARACTERISTICS OF MALIGNANT IN CULTURE
CELLS
Although the conceptsof monoclonality , tumor stem cells, tumor heterogeneity, and invasiveness have primarily been developed by observing tu-
TUMOR
5
CELL GROWTH
mors growing in vivo, an understandingof the molecular mechanisms underlying the abnormal growth of cancer cells has been established by propagating malignant cells in culture. From this research, we have learned that the growth properties of cancer cells in culture differ from normal cells in the following characteristics: (1) cell growth control; (2) cell morphology; (3) densitydependentinhibition of growth; (4) anchoragedependence;(5) contact inhibition of growth; and (6) adhesiveness(Table 1). ‘M
Receptor
B
Cell Growth Control Proliferative life span. Normal cell growth and differentiation in culture depend on the presence of growth factors that stimulate cell proliferation. In normal cells, growth factors produced by one cell type bind to specific receptorson the cell membraneof the target cell, initiating a series of events that lead to mitosis. lo In malignant tumors, cells continue to proliferate becauseof a decrease in growth factor requirements by tumor cells and changes in the way the tumor cells interact with growth factors.9-‘3 Decreased growth factor requirements. Because transformed cells are not as dependent on hormone and growth factor requirementsasnormal cells, they proliferate in culture when concentrations of growth factors are much lower than those Table 1. In Vitro
Growth Characteristic
Normal
Cell growth Proliferative life span Growth factor requirements Morphology Shaoe
Nuclear-tocytoplasm Mitoses Nucleus
Growth Characteristics Verus Tumor Cells
ratio
Densitydependent inhibition of growth Anchorage dependence Contact inhibition of movement Adhesiveness
Cell
of Normal
Tumor
Cell
Finite
Infinite
High
Low or Absent
Regular in size and shape; flat
Irregular in size and shape; pleomorphic
Low Rare Single; Euploid DNA content
High Frequent Multiple; Euploid or aneuploid DNA content
Present Present
Absent Absent
Present High
Absent Low
Fig 2. (A) Schematic illustration of the action of growth factors to stimulate cell proliferation in normal cells. (61 Possible changes in growth factor-related pathways that might lead to malignant transformation: (1) production of growth factors and autostimulation; (2) production of factors that stimulate activation of the receptor; (3) oonstitutive activetion of the intracellular regulatory mechanism. (Reprinted with permission.‘)
required by normal cells. It has also been demonstrated that when the concentration of certain growth-control substancesfalls below a certain threshold level, normal cells go into quiescenceor a resting phase,whereastransformedcells lack this ability to stop their proliferation in response to lowered nutrient concentration.l2 Changes in interaction with cell growth factors. Transformedcells are known to interact differently with growth factors than do normal cells. Thesedifferencescan be explained by severalpossibilities: The transformed cell (1) produces its own growth factors; (2) responds more continuously or efficiently to growth factors produced outside the cell; and/or (3) does not respond to the substancesthat regulate continued proliferation. lo-l3 Figure 2 summarizesthe differences in interactions of growth factors with normal cells versus transformedcells. Cell Morphology
Each human cell is made up of the cell membrane, also called the plasma membrane;the cellular fluid and subcellular structures (organelles) enclosed in the cytoplasm; and the nucleus, the home of the cell’s genetic material. In normal cells, the nucleus representsonly a small percent-
6
JULENA
age of the entire cell, with the cellular fluid and subcellular structures in the cytoplasm comprising the majority of the cell space. The appearanceof cancer cells is significantly different than their normal counterparts (Fig 3). r4 Cancer cells have a higher nucleus-to-cytoplasm ratio, prominent nucleoli, many mitosesevidenced by large amounts of chromatin material, and relatively little specialized structure.’ Density-Dependent inhibition
of Growth
LIND
cells; consequently,when two cells come into contact, one or both of them will stop and move in anotherdirection. This ensuresthat the cells do not overlap. This characteristicis referred to as contact inhibition of movement. Transformed cells lack contact inhibition of movementand they passover or under each other, growing on top of one another.6 Adhesiveness
As normal cells in a laboratory culture dish proliferate and becomemore crowded, they align and pack into their spacein a single layer, overlapping only at the cell borders. Once the cells reach a finite density that conforms to their space, they ceaseproliferation. Transformedcells do not cease proliferation but continue to replicate to much higher densities and appear crisscrossedand chaotic in their growth.‘9’5
Related to the characteristic of loss of contact inhibition is the fact that tumor cells are less adhesive than normal cells and are less firmly attached to neighboring cells or the surface of the culture dish.’ Loss of adhesivenessand loss of contact inhibition contribute to the metastatic potential of tumor cells. r6
Anchorage Dependence
A key characteristic of transformedcells is that they continue to proliferate under circumstancesin which normal cells ceaseproliferation. In animals or in culture, cells are either proliferating or quiescent. Tumors grow becausethey contain a population of cells that is expanding as a result of cell division. They differ from normal tissues because the population of tumor cells does not respond effectively to the homeostatic control mechanisms that maintain the appropriate number of cells in normal renewal tissues.
Most normal cells need contact with substratum in the extracellular environment to reproduce and to keep from moving haphazardly. Transformed cells grow without attachment to a substratum. This can be seenby their ability to form colonies when suspendedas single cells in agar.6 Contact Inhibition
of Movement
When normal cells are placed in culture, they have the ability to respondto the presenceof other
CELL GROWTH CONTROL
Fig 3. Artist’s concaption of microscopic apfmaranw of normal (A) versus transformed IB) cells. (Reprinted with permission.‘9
TUMOR
7
CELL GROWTH
The Cell Cycle
Cell proliferation involves two easily recognized and coordinated events: the duplication of DNA and the physical division into two daughter cells, called mitosis. The process of cell replication, protein and DNA synthesis, and division is called the “cell cycle” (Fig 4). The phases in a cell’s cycle are known as G, which is the resting (quiescent)state; G,, a phasein which protein synthesistakes place in preparation for the S phase of DNA synthesis; and G,, where further protein synthesistakesplace in preparation for the M phaseduring which mitosis takes place. Human cells that are actively dividing, such as the epithelial cells that line the inside of the gastrointestinal tract, are rarely in the resting state. On the other hand, nerve cells are almost always in G,. It has been shown that an RNA polymerase directs the entry of resting cells into S phase.” Cell Kinetics
A completecell cycle for all the cells in a cancer clone is referred to as a “doubling” becausethe number of cells doubles. The meancell-cycle time in human tumors is often short, approximately 2 to 3 days, but becausenot all cells are in cycle at the sametime, the observed doubling time is usually much longer. Exponential growth often has mean volume doubling times of 2 to 3 months for com-
Fig 4. The cell cycle, indicating the periods of interphasa (0,). DNA synthesis (S), the pariod between the end of DNA synthesis, and the beginning of mitosis interval (MI. The GO state is also depicted as a side extension of interphase, with the possibility that the GO cell may raenter the cycle (dashed line). (Reprinted with permission.‘4)
mon types of human cancers.” Tumors probably have undergone approximately 30 doublings from a single cell before they are clinically detected. If this theory of growth rate is accepted, then the tumor would be expected to be lethal in 7 to 10 doublings after detection. Two research techniques, thymidine autoradiography and flow cytometry, have led to detailed studiesof the cell cycle and cell proliferation in normal cells and in cancer cells.18 A quiescent or resting cell is one that is not increasing in mass or passing through the cell cycle. Only a single step is under tight control; the commitment in Gi to go through S. The inputs for this step are poorly understood, but they involve whether the cell has grown to a sufficient size to warrant dividing into two and whether the nutritional stateof the cells is above a fixed threshold.l9 This processappearsto be controlled by the concentration of a protein called cyclin. Cyclins build up in the cell in interphase and then are degraded during mitosis. If cells are deprived of cyclin, they will remain blocked in a G,-like state.18 TREATMENT IMPLICATIONS
An understandingof tumor growth and cell kinetics is the foundation of many aspectsof cancer treatment. Radiotherapy
Ionizing radiation causes damage to cells and tissuesas a result of the deposit and absorption of energy and ionization. The biological effects take place at the individual cell level although the consequencescan be seenthroughout the body.*’ Radiation works by ionizing molecules inside the cell and causing damage. The effect at the cellular level can either be direct or indirect. Becausethe ability to continue to proliferate is one of the most radiosensitive functions of the cell, interfering with proliferation is the prime target of radiation therapy. Therefore, although radiation can cause damageto any molecule in a cell, it is thought that the damageto DNA is the most crucial in relation to cell death. A direct hit occurs when a DNA molecule inside the cell is damaged.*l Many factors affect a cell’s responseto radiation. The amount of renewal or proliferation, the location of the tumor, the amount of oxygen available within tumor cells, and the position of the cell
8 in the cell cycle are all thought to influence radiotherapy effectiveness.*’ Oxygen is also an important factor as a radiation sensitizer. Tumors that are well oxygenated show a greater response to radiation therapy. The oxygen effect can be expressed as the oxygen enhancement ratio (OER). Applying the concept of cell kinetics described above implies that the larger the tumor (ie, the greater number of doublings) the greater the tumor bulk, and thus the greater likelihood of hypoxic cells. Following that model, a larger tumor is more difficult to treat than a smaller tumor because of its poor oxygenation and relative radioresistance. ** Fractionation of radiotherapy is based on cell kinetic concepts. Radiation therapy for cancer usually involves giving 20 to 30 individual dose fractions of 2 to 2.5 Gy spread over a period of 4 to 6 weeks. Because cancer cells are not able to repair themselves as effectively or rapidly as normal cells, this treatment strategy allows normal cells to renew themselves. Chemotherapy Curative treatment for cancer is aimed at killing stem cells responsible for the neoplastic clone. A logical application of an understanding of the growth of tumor cells has been to try to identify tumor-specific stem cells and then to determine which cytotoxic drug is most effective against the tumor. Many investigators have tried to increase the efficiency of chemotherapy through the use of assays to assessthe response of the tumor clone to specific chemotherapy agents (clonogenic assays).23 Unfortunately the practical use of the assays for guiding individual treatment has been limited. The role of tumor growth and kinetics is important to understanding the action of cytotoxic therapy. Chemotherapy agents usually work best on cells that are actively proliferating. The drugs are categorized into groups (such as alkylating agents, antimetabolites , vinca alkaloids, and antibiotics) according to their mechanism of action on those proliferating cells. For example, certain chemotherapy drugs (the antimetabolites) interfere with DNA synthesis in the S phase. Other drugs, such as the vinca alkaloids, interfere with spindle construction during mitosis. The alkylating agents, such as cyclophosphamide and nitrogen mustard, have similar phase activity to that observed in radiation therapy, with
JULENA
LIND
two peaks of maximum lethal activity, one in G, to M phase, and one near the G,/S phase boundary. Cisplatin probably has greater activity for some cells in G,, with little or no phase specificity for others. Finally, the antibiotics are active in all phases of the cell cycle.24,25 The concept of exponential growth plays a role in chemotherapy treatment as well as in radiation therapy. As the tumor doubles and grows larger, the rate of proliferation slows. Because chemotherapy is thought to be most effective against cells that are actively dividing, it becomes more difficult to treat large tumors that have slowed proliferation rates. 24 It is also likely that after chemotherapy treatment, the actively dividing cancer cells that are responsive to chemotherapy are killed off, and the remaining cells are part of a resistant neoplastic clone of stem cells.26 Other Treatment Implications Understanding the cell cycle and the length of time it takes for a cell to pass through a cycle, or “double” itself, has increased our understanding of how certain kinds of tumors should be treated and what to expect from their natural history. Today, the advent of genetic cloning and mapping, along with other biotechnologies, has opened a new world of treatment potentials. For example, understanding growth factors of white blood cells and being able to “genetically” reproduce them has led to the production of colony-stimulating factors (CSF). In general, CSFs are naturally occurring glycoproteins that target either the granulocyte (G-CSF) or the macrophage (GM-CSF) cell lines to stimulate their growth. The two factok have slightly different therapeutic implications. G-CSF stimulates the activation of neutrophils whereas GM-CSF has a broader capability to stimulate granulocytes, monocytes , and macrophages . Studies have shown that CSFs have enabled patients to undergo extremely aggressive curative chemotherapy without seriously jeopardizing their immune status.27 The reader is referred to a review of various biotechnological therapies and nurses’ roles in caring for patients receiving such therapies.28 The interleukins are another class of cellular regulatory substances that have therapeutic implications. Interleukin 2 (IL-2) is a glycoprotein produced by helper T cells after stimulation. Its cellular effect is to bind with receptors on T and other lymphocytes, and it appears to be an essential factor in the growth of T cells. IL-2 combined with
TUMOR CELL GROWTH
9
lymphokine-activated killer cells (LAK) has resulted in a regressionof metastasesin many tumors including bladder cancer, sarcoma, melanoma, and adenocarcinomasof the colon.29 SUMMARY
Cancer cells differ from normal cells in many ways, but most importantly by not responding to normal growth-control mechanisms. Whereasthe growth and division of normal cells is carefully
regulated to meet the needs of the body, tumor cells proliferate autonomously and continually, eventually interfering with and destroying the functions of normal tissue. Knowledge of molecular cell biology has grown exponentially over the last decade. Yet much remains to be understoodbefore there can be a significant impact on our ability to design more effective therapeutic strategies for cancer patients, thereby decreasingmortality.
REFERENCES 1. Buick RN, Tannock IF: Propertiesof malignant cells, in Tannock IF, Hill RP (eds): The Basic Science of Oncology. New York, NY, Pergamon, 1987, pp 127-139 2. Hill RP, Tannock IF: Introduction: Cancer as a cellular disease, in Tannock IF, Hill RP (eds): The Basic Science of Oncology. New York, NY, Pergamon, 1987, pp l-4 3. Spremulli EN, Dexter DL: Human tumor cell heterogeneity and metastasis.J Clin Oncol 1:496-509, 1983 4. Heppner GH: Tumor heterogeneity. CancerRes44:22592265, 1984 5. Fidler Ll, Hart IR: Biologic diversity in metastatic neoplasms: Origins and implications. Science217998-1003, 1982 6. Skehan P, Friedman SJ: Malignant transformation: In vitro methodsand in vivo correlates, in Cameron IL, Pool TB (eds): The Transformed Cell. New York, NY, Academic, 1981, pp 8-66 7. Greenberg PD: Mechanisms of tumor immunology, in Stites DP, Terr AI (eds) Basic and Clinical Immunology. Norwalk, CT, Appleton & Lange, 1991, pp 580-587 8. Minden MD: Oncogenes,In Tannock IF, Hill RP (eds): The Basic Science of Oncology. New York, NY, Pergamon, 1987, pp 72-88 9. Burck KB, Liu ET, Lanick JW: Oncogenes:An Introduction to the Concept of Cancer. New York, NY, SpringerVerlag, 1988 10. WatsonJD, Hopkins NH, Roberts JW, et al: Molecular Biology of the Gene, Vol2. Specialized Aspects (ed 4). Menlo Park, CA, Benjamin/Cummings, 1987, pp 747-l 163 11. Heldin CH, WestermarkB: Growth factors: Mechanism of action and relation to oncogenes.Cell 37:9-20, 1984 12. Watertield MD: The role of growth factors in cancer, in Franks LM, Teich NM (eds). Introduction to the Cellular and Molecular Biology of Cancer. Oxford, England, Oxford University Press, 1986, pp 27-39 13. Weinberg RA (ed): Oncogenesand the Molecular Origins of Cancer. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1989 14. Pitot HC: Fundamentalsof Oncology. New York, Marcell Dekker, 1986 15. Abercrombie M, Heaysman JBM: Observations on the social behaviour of cells in tissue culture. II. “Monolayering” of tibroblasts. Exp Cell Res 6293-306, 1954
16. Killion JJ, Fidler IJ: The biology of tumor metastasis. Semin Oncol 16:106-115,1989 17. TannockIF: Tumor growth and cell kinetics, in Tannock IF, Hill RP (eds): The Basic Scienceof Oncology. New York, NY, Pergamon, 140-159, 1987 18. Dame11J, Lodish H, Baltimore D: Molecular Cell Biology. New York, NY, Scientific American Books, 1990 19. Cory JG, Szentivanyi A: CancerBiology and Therapeutics. New York, NY, Plenum, 1986 20. Hill Rp: Cellular basis of radiotherapy, in Tannock IF, Hill RP (eds):The Basic Scienceof Oncology. New York, NY, Pergamon,237-255, 1987 21. Hall ET, Cox JD (eds): Radiation Oncology: Rationale, Techniquesand Results. St. Louis, MO, Mosby, 1989,pp l-57 22. Withers HR: Biologic basis of radiation therapy, in Perez CA, Brady LW (eds): Principles and Practice of Radiation Oncology. Philadelphia, PA, Lippincott, 1987, pp 67-98 23. Hamburger AW, Salmon SE: Primary bioassay of human tumor stem cells. Science 197:461-463,1977 24. Tannock IF: Biologic properties of anticancerdrugs, in Tannock IF, Hill RP (eds): The Basic Science of Oncology. New York, NY, Pergamon, 1987, pp 278-291 25. Skipper HE, Schabel FM, Wilcox WS: Experimental evaluation of potential anticanceragents. XIII. On the criteria and kinetics associatedwith “curability” of experimental leukemia. Cancer ChemotherRep 35:l-l 11, 1964 26. Goldie JH, Coldman AJ: A mathematicalmodel for relating the drug sensitivity of tumors to their spontaneousmutation rate. Cancer Treat Rep 63:1727-1733, 1979 27. JassakPF: Biotherapy, in Groenwald SL, Frogge MH, GoodmanM, Yarbro CH (eds): Cancer Nursing Principles and Practice. Boston, MA, Jones& Bartlett, 1990, pp 284-306 28. Antman KS, Griffen JD, Elias AS, et al: Effect of recombinant human granulocyte-macrophagecolony-stimulating factor on chemotherapyinduced myelosuppression.N Engl J Med 319593-598, 1988 29. RosenbergSA, Lotze MT, Mueel LM, et al: Observations on the systematic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 313:1485-1492, 1985
and Cell Kinetics
Julena Lind
N UNDERSTANDING of cancer requires a basic knowledge of the differences between normal and malignant cells. Understandingnormal cell structure, growth, and kinetics through the life cycle is the critical first step in that knowledge. This article presents the characteristics of malignant cells, in vivo and in vitro, tumor growth and kinetics, and finally a summary of the biology underlying cancer treatment.
A
IN WV0 CHARACTERISTICS
OF CANCER CELLS
Most of the understandingof cancer cell growth has been achieved by changing normal cells into malignant cells outside of the human body. In order to apply this research to human beings, we must know how the behavior of cells in vitro applies to the behavior of cells in vivo. Increased Cell Renewal And Unresponsiveness to Growth Controls
Cell growth is a carefully regulated processthat respondsto specific needsof the body. When the controls that regulate cell replication break down, a cell begins to grow and divide without regard to the needfor more cells of its type. The descendants of such a cell inherit the propensity to replicate, and the result may be a clone of cells able to expand indefinitely. Most human tumors arise in renewing cell populations or in tissue that has been exposedto a chronic irritation that causesproliferation. Stem Cells
In normal tissues, new cells are derived from a small number of stem cells that have a high capacity for cell proliferation, but their actual rate of cell division is usually quite low unless there is tissue injury or demand. Stem cells have two critical functions: to generate a large family of descendants that will perform the function of the tissue and to renew themselvesso that a stable number of stem cells remain (Fig 1). i Monoclonality
and Tumor Heterogeneity
Tumors are thought to be monoclonal: that is, they arise from the transformation of a single precursor cell, which then proliferates to form a neoplastic clone. Transformation is most often thought Seminars in Oncology Nursing, Vol8, No 1 (February), 1992: pp 3-g
to be a result of carcinogenic events occurring in a single stem cell.2 However, when individual cells within tumors are studied, it is found that the tumor is comprisedof subpopulationsof biologically diverse cells that have differentiated from the same stem cell clone. The biological variability of cells within a tumor is referred to as tumor heterogeneity.374 Invasiveness
Most tumors pose little risk to humans because they are localized. Benign tumors contain cells that very closely resemble normal cells and, in fact, function like normal cells. Ordinarily, these benign tumors, like normal cells in tissues, remain localized in the appropriatetissues. However, malignant tumors are characterizedby their invasiveness and ability to spread throughout the body. They contain cells that are usually less well differentiated than normal cells, and their biological properties change over time. Changes in Cell-to-Cell Interaction
Invasiveness and metastasis are the result of changes in cell-to-cell interactions. Normal cells are restrictedto certain organsor tissuesbecauseof cell-to-cell recognition or by the presenceof physical barriers such as the extracellular matrix. Cells recognizeeachother by processesthat take place on the surface of the cell. These processes dictate which cells bind together and interact.’ Normal cells recognize and respectthe boundaries and the territory of the cell surrounding them. In cancer cells, surfaceenzymesare altered and recognition is impaired resulting in a number of biological changes including loss of contact inhibition. ’ The response to physical barriers is also changed;for example, many metastatictumor cells From the Department of Nursing, University of Southern California, Los Angeles, CA. Julena Lind, MS, RN: Interim Chair, Department of Nursing, University of Southern California. Address reprint requests to Julena Lind, MS, RN, 320 West 15th St, Los Angeles, CA 90015. Copyright 0 1992 by W.B. Saunders Company 0749-2081192/0801-0002$5.OOlO
3
JULENA
Molurolion without division
Functional finite
cells
with
lifetime
have the ability to “digest” their way through extracellular matrices by secretion of proteolytic enzymes or proteases.6 Escape From Immune Surveillance
Another recognized property of malignant cells in vivo is their ability to elude the immune system. It might be expected that the alterations in cell structure associatedwith malignancy would lead the immune system to recognize tumor cells as foreign. The blood even contains a “natural killer” cell that can recognize and kill many types of tumor cells while sparing normal cells. Despite this, tumor cells find ways to elude immune detection.’ Appelbaum describes this phenomenon elsewherein this issue. ONCOGENES
It is now clear that genetic changesare responsible for neoplastic transformation. Research in molecular biology has been focused largely on identifying the genesthat play a causal role in tumor development. Genesinvolved in the induction of neoplastic transformation were first identified in tumor viruses and later in human tumors. Oncogenesare genesthat promote growth; their protein products “turn on” cell division. When normal growth genes mutate to form oncogenes, their abnormal protein products may lead to tumor growth.’
LIND
Fig 1. The hierarchy of cells in renewal tissue. The mowlation is maintained by clonal xpansion from a small number of stem cells. (Reprinted with permission.‘)
Most oncogenesare derived from normal cellular genescalled proto-oncogenes.Proto-oncogenes are not cancer-causingin their normal cellular environment, and most of them presumably fulfill essentialphysiological functions in cell growth and development. The transition from normal genetic material, a proto-oncogene, to an oncogene includes changesin gene control and gene expression, but it is not yet clear exactly which changes in structure and expression are necessary.Not all human tumors contain identifiable oncogenes.9 Oncogenes and Tumor Growth
Some experiments have linked growth factors and oncogenesand suggesta relationship between malignancy and the loss of mechanismsthat control cell proliferation in normal tissues.lo-‘* Although growth control is not completely understood, there are at least three types of proteins that participate in the process:growth factors, growth factor receptors found on the plasma membrane, and intracellular proteins. Oncogenesand growth control factors are discussedin detail by Yarbro elsewherein this issue. CHARACTERISTICS OF MALIGNANT IN CULTURE
CELLS
Although the conceptsof monoclonality , tumor stem cells, tumor heterogeneity, and invasiveness have primarily been developed by observing tu-
TUMOR
5
CELL GROWTH
mors growing in vivo, an understandingof the molecular mechanisms underlying the abnormal growth of cancer cells has been established by propagating malignant cells in culture. From this research, we have learned that the growth properties of cancer cells in culture differ from normal cells in the following characteristics: (1) cell growth control; (2) cell morphology; (3) densitydependentinhibition of growth; (4) anchoragedependence;(5) contact inhibition of growth; and (6) adhesiveness(Table 1). ‘M
Receptor
B
Cell Growth Control Proliferative life span. Normal cell growth and differentiation in culture depend on the presence of growth factors that stimulate cell proliferation. In normal cells, growth factors produced by one cell type bind to specific receptorson the cell membraneof the target cell, initiating a series of events that lead to mitosis. lo In malignant tumors, cells continue to proliferate becauseof a decrease in growth factor requirements by tumor cells and changes in the way the tumor cells interact with growth factors.9-‘3 Decreased growth factor requirements. Because transformed cells are not as dependent on hormone and growth factor requirementsasnormal cells, they proliferate in culture when concentrations of growth factors are much lower than those Table 1. In Vitro
Growth Characteristic
Normal
Cell growth Proliferative life span Growth factor requirements Morphology Shaoe
Nuclear-tocytoplasm Mitoses Nucleus
Growth Characteristics Verus Tumor Cells
ratio
Densitydependent inhibition of growth Anchorage dependence Contact inhibition of movement Adhesiveness
Cell
of Normal
Tumor
Cell
Finite
Infinite
High
Low or Absent
Regular in size and shape; flat
Irregular in size and shape; pleomorphic
Low Rare Single; Euploid DNA content
High Frequent Multiple; Euploid or aneuploid DNA content
Present Present
Absent Absent
Present High
Absent Low
Fig 2. (A) Schematic illustration of the action of growth factors to stimulate cell proliferation in normal cells. (61 Possible changes in growth factor-related pathways that might lead to malignant transformation: (1) production of growth factors and autostimulation; (2) production of factors that stimulate activation of the receptor; (3) oonstitutive activetion of the intracellular regulatory mechanism. (Reprinted with permission.‘)
required by normal cells. It has also been demonstrated that when the concentration of certain growth-control substancesfalls below a certain threshold level, normal cells go into quiescenceor a resting phase,whereastransformedcells lack this ability to stop their proliferation in response to lowered nutrient concentration.l2 Changes in interaction with cell growth factors. Transformedcells are known to interact differently with growth factors than do normal cells. Thesedifferencescan be explained by severalpossibilities: The transformed cell (1) produces its own growth factors; (2) responds more continuously or efficiently to growth factors produced outside the cell; and/or (3) does not respond to the substancesthat regulate continued proliferation. lo-l3 Figure 2 summarizesthe differences in interactions of growth factors with normal cells versus transformedcells. Cell Morphology
Each human cell is made up of the cell membrane, also called the plasma membrane;the cellular fluid and subcellular structures (organelles) enclosed in the cytoplasm; and the nucleus, the home of the cell’s genetic material. In normal cells, the nucleus representsonly a small percent-
6
JULENA
age of the entire cell, with the cellular fluid and subcellular structures in the cytoplasm comprising the majority of the cell space. The appearanceof cancer cells is significantly different than their normal counterparts (Fig 3). r4 Cancer cells have a higher nucleus-to-cytoplasm ratio, prominent nucleoli, many mitosesevidenced by large amounts of chromatin material, and relatively little specialized structure.’ Density-Dependent inhibition
of Growth
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cells; consequently,when two cells come into contact, one or both of them will stop and move in anotherdirection. This ensuresthat the cells do not overlap. This characteristicis referred to as contact inhibition of movement. Transformed cells lack contact inhibition of movementand they passover or under each other, growing on top of one another.6 Adhesiveness
As normal cells in a laboratory culture dish proliferate and becomemore crowded, they align and pack into their spacein a single layer, overlapping only at the cell borders. Once the cells reach a finite density that conforms to their space, they ceaseproliferation. Transformedcells do not cease proliferation but continue to replicate to much higher densities and appear crisscrossedand chaotic in their growth.‘9’5
Related to the characteristic of loss of contact inhibition is the fact that tumor cells are less adhesive than normal cells and are less firmly attached to neighboring cells or the surface of the culture dish.’ Loss of adhesivenessand loss of contact inhibition contribute to the metastatic potential of tumor cells. r6
Anchorage Dependence
A key characteristic of transformedcells is that they continue to proliferate under circumstancesin which normal cells ceaseproliferation. In animals or in culture, cells are either proliferating or quiescent. Tumors grow becausethey contain a population of cells that is expanding as a result of cell division. They differ from normal tissues because the population of tumor cells does not respond effectively to the homeostatic control mechanisms that maintain the appropriate number of cells in normal renewal tissues.
Most normal cells need contact with substratum in the extracellular environment to reproduce and to keep from moving haphazardly. Transformed cells grow without attachment to a substratum. This can be seenby their ability to form colonies when suspendedas single cells in agar.6 Contact Inhibition
of Movement
When normal cells are placed in culture, they have the ability to respondto the presenceof other
CELL GROWTH CONTROL
Fig 3. Artist’s concaption of microscopic apfmaranw of normal (A) versus transformed IB) cells. (Reprinted with permission.‘9
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CELL GROWTH
The Cell Cycle
Cell proliferation involves two easily recognized and coordinated events: the duplication of DNA and the physical division into two daughter cells, called mitosis. The process of cell replication, protein and DNA synthesis, and division is called the “cell cycle” (Fig 4). The phases in a cell’s cycle are known as G, which is the resting (quiescent)state; G,, a phasein which protein synthesistakes place in preparation for the S phase of DNA synthesis; and G,, where further protein synthesistakesplace in preparation for the M phaseduring which mitosis takes place. Human cells that are actively dividing, such as the epithelial cells that line the inside of the gastrointestinal tract, are rarely in the resting state. On the other hand, nerve cells are almost always in G,. It has been shown that an RNA polymerase directs the entry of resting cells into S phase.” Cell Kinetics
A completecell cycle for all the cells in a cancer clone is referred to as a “doubling” becausethe number of cells doubles. The meancell-cycle time in human tumors is often short, approximately 2 to 3 days, but becausenot all cells are in cycle at the sametime, the observed doubling time is usually much longer. Exponential growth often has mean volume doubling times of 2 to 3 months for com-
Fig 4. The cell cycle, indicating the periods of interphasa (0,). DNA synthesis (S), the pariod between the end of DNA synthesis, and the beginning of mitosis interval (MI. The GO state is also depicted as a side extension of interphase, with the possibility that the GO cell may raenter the cycle (dashed line). (Reprinted with permission.‘4)
mon types of human cancers.” Tumors probably have undergone approximately 30 doublings from a single cell before they are clinically detected. If this theory of growth rate is accepted, then the tumor would be expected to be lethal in 7 to 10 doublings after detection. Two research techniques, thymidine autoradiography and flow cytometry, have led to detailed studiesof the cell cycle and cell proliferation in normal cells and in cancer cells.18 A quiescent or resting cell is one that is not increasing in mass or passing through the cell cycle. Only a single step is under tight control; the commitment in Gi to go through S. The inputs for this step are poorly understood, but they involve whether the cell has grown to a sufficient size to warrant dividing into two and whether the nutritional stateof the cells is above a fixed threshold.l9 This processappearsto be controlled by the concentration of a protein called cyclin. Cyclins build up in the cell in interphase and then are degraded during mitosis. If cells are deprived of cyclin, they will remain blocked in a G,-like state.18 TREATMENT IMPLICATIONS
An understandingof tumor growth and cell kinetics is the foundation of many aspectsof cancer treatment. Radiotherapy
Ionizing radiation causes damage to cells and tissuesas a result of the deposit and absorption of energy and ionization. The biological effects take place at the individual cell level although the consequencescan be seenthroughout the body.*’ Radiation works by ionizing molecules inside the cell and causing damage. The effect at the cellular level can either be direct or indirect. Becausethe ability to continue to proliferate is one of the most radiosensitive functions of the cell, interfering with proliferation is the prime target of radiation therapy. Therefore, although radiation can cause damageto any molecule in a cell, it is thought that the damageto DNA is the most crucial in relation to cell death. A direct hit occurs when a DNA molecule inside the cell is damaged.*l Many factors affect a cell’s responseto radiation. The amount of renewal or proliferation, the location of the tumor, the amount of oxygen available within tumor cells, and the position of the cell
8 in the cell cycle are all thought to influence radiotherapy effectiveness.*’ Oxygen is also an important factor as a radiation sensitizer. Tumors that are well oxygenated show a greater response to radiation therapy. The oxygen effect can be expressed as the oxygen enhancement ratio (OER). Applying the concept of cell kinetics described above implies that the larger the tumor (ie, the greater number of doublings) the greater the tumor bulk, and thus the greater likelihood of hypoxic cells. Following that model, a larger tumor is more difficult to treat than a smaller tumor because of its poor oxygenation and relative radioresistance. ** Fractionation of radiotherapy is based on cell kinetic concepts. Radiation therapy for cancer usually involves giving 20 to 30 individual dose fractions of 2 to 2.5 Gy spread over a period of 4 to 6 weeks. Because cancer cells are not able to repair themselves as effectively or rapidly as normal cells, this treatment strategy allows normal cells to renew themselves. Chemotherapy Curative treatment for cancer is aimed at killing stem cells responsible for the neoplastic clone. A logical application of an understanding of the growth of tumor cells has been to try to identify tumor-specific stem cells and then to determine which cytotoxic drug is most effective against the tumor. Many investigators have tried to increase the efficiency of chemotherapy through the use of assays to assessthe response of the tumor clone to specific chemotherapy agents (clonogenic assays).23 Unfortunately the practical use of the assays for guiding individual treatment has been limited. The role of tumor growth and kinetics is important to understanding the action of cytotoxic therapy. Chemotherapy agents usually work best on cells that are actively proliferating. The drugs are categorized into groups (such as alkylating agents, antimetabolites , vinca alkaloids, and antibiotics) according to their mechanism of action on those proliferating cells. For example, certain chemotherapy drugs (the antimetabolites) interfere with DNA synthesis in the S phase. Other drugs, such as the vinca alkaloids, interfere with spindle construction during mitosis. The alkylating agents, such as cyclophosphamide and nitrogen mustard, have similar phase activity to that observed in radiation therapy, with
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two peaks of maximum lethal activity, one in G, to M phase, and one near the G,/S phase boundary. Cisplatin probably has greater activity for some cells in G,, with little or no phase specificity for others. Finally, the antibiotics are active in all phases of the cell cycle.24,25 The concept of exponential growth plays a role in chemotherapy treatment as well as in radiation therapy. As the tumor doubles and grows larger, the rate of proliferation slows. Because chemotherapy is thought to be most effective against cells that are actively dividing, it becomes more difficult to treat large tumors that have slowed proliferation rates. 24 It is also likely that after chemotherapy treatment, the actively dividing cancer cells that are responsive to chemotherapy are killed off, and the remaining cells are part of a resistant neoplastic clone of stem cells.26 Other Treatment Implications Understanding the cell cycle and the length of time it takes for a cell to pass through a cycle, or “double” itself, has increased our understanding of how certain kinds of tumors should be treated and what to expect from their natural history. Today, the advent of genetic cloning and mapping, along with other biotechnologies, has opened a new world of treatment potentials. For example, understanding growth factors of white blood cells and being able to “genetically” reproduce them has led to the production of colony-stimulating factors (CSF). In general, CSFs are naturally occurring glycoproteins that target either the granulocyte (G-CSF) or the macrophage (GM-CSF) cell lines to stimulate their growth. The two factok have slightly different therapeutic implications. G-CSF stimulates the activation of neutrophils whereas GM-CSF has a broader capability to stimulate granulocytes, monocytes , and macrophages . Studies have shown that CSFs have enabled patients to undergo extremely aggressive curative chemotherapy without seriously jeopardizing their immune status.27 The reader is referred to a review of various biotechnological therapies and nurses’ roles in caring for patients receiving such therapies.28 The interleukins are another class of cellular regulatory substances that have therapeutic implications. Interleukin 2 (IL-2) is a glycoprotein produced by helper T cells after stimulation. Its cellular effect is to bind with receptors on T and other lymphocytes, and it appears to be an essential factor in the growth of T cells. IL-2 combined with
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lymphokine-activated killer cells (LAK) has resulted in a regressionof metastasesin many tumors including bladder cancer, sarcoma, melanoma, and adenocarcinomasof the colon.29 SUMMARY
Cancer cells differ from normal cells in many ways, but most importantly by not responding to normal growth-control mechanisms. Whereasthe growth and division of normal cells is carefully
regulated to meet the needs of the body, tumor cells proliferate autonomously and continually, eventually interfering with and destroying the functions of normal tissue. Knowledge of molecular cell biology has grown exponentially over the last decade. Yet much remains to be understoodbefore there can be a significant impact on our ability to design more effective therapeutic strategies for cancer patients, thereby decreasingmortality.
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16. Killion JJ, Fidler IJ: The biology of tumor metastasis. Semin Oncol 16:106-115,1989 17. TannockIF: Tumor growth and cell kinetics, in Tannock IF, Hill RP (eds): The Basic Scienceof Oncology. New York, NY, Pergamon, 140-159, 1987 18. Dame11J, Lodish H, Baltimore D: Molecular Cell Biology. New York, NY, Scientific American Books, 1990 19. Cory JG, Szentivanyi A: CancerBiology and Therapeutics. New York, NY, Plenum, 1986 20. Hill Rp: Cellular basis of radiotherapy, in Tannock IF, Hill RP (eds):The Basic Scienceof Oncology. New York, NY, Pergamon,237-255, 1987 21. Hall ET, Cox JD (eds): Radiation Oncology: Rationale, Techniquesand Results. St. Louis, MO, Mosby, 1989,pp l-57 22. Withers HR: Biologic basis of radiation therapy, in Perez CA, Brady LW (eds): Principles and Practice of Radiation Oncology. Philadelphia, PA, Lippincott, 1987, pp 67-98 23. Hamburger AW, Salmon SE: Primary bioassay of human tumor stem cells. Science 197:461-463,1977 24. Tannock IF: Biologic properties of anticancerdrugs, in Tannock IF, Hill RP (eds): The Basic Science of Oncology. New York, NY, Pergamon, 1987, pp 278-291 25. Skipper HE, Schabel FM, Wilcox WS: Experimental evaluation of potential anticanceragents. XIII. On the criteria and kinetics associatedwith “curability” of experimental leukemia. Cancer ChemotherRep 35:l-l 11, 1964 26. Goldie JH, Coldman AJ: A mathematicalmodel for relating the drug sensitivity of tumors to their spontaneousmutation rate. Cancer Treat Rep 63:1727-1733, 1979 27. JassakPF: Biotherapy, in Groenwald SL, Frogge MH, GoodmanM, Yarbro CH (eds): Cancer Nursing Principles and Practice. Boston, MA, Jones& Bartlett, 1990, pp 284-306 28. Antman KS, Griffen JD, Elias AS, et al: Effect of recombinant human granulocyte-macrophagecolony-stimulating factor on chemotherapyinduced myelosuppression.N Engl J Med 319593-598, 1988 29. RosenbergSA, Lotze MT, Mueel LM, et al: Observations on the systematic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 313:1485-1492, 1985
