chapter 3 The Technologies - Princeton

chapter 3

The Technologies

“We must, as far as we can, isolate physiological occurrences outside the organism by meansof experimental procedures. This isolation allows us to see and understand better the deepestassociations of the phenomenon, so that their vital role maybe followed later in the organism. ”

—CIaude Bernard1813-1878

CONTENTS

PageTissue and Cell Culture Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Culturing Human Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Human Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Using Cell Cultures .. <. .. .. .. .. ...4 ... .....$ . . . . . . . . . . . . . . . . . . . . . . . . .

Hybridoma Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Monoclinal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Lymphokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Recombinant DNA Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Gene Cloning . . . . . . . . . . . . . . ..t,,.... .......... .......... . . . . . . . . . . . .

Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Chapter

Table No.

3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

313233353537384141414445

3. Comparison of Microbial and Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 314. Some Nutrient and Growth Condition Requirements for Culturing Human

Cells .. .. .. .. .. .. .. .. . . $ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.Some Lymphokines With Therapeutic Potential . . . . . . . . . . . . . .

FiguresF@reNo.

3. Plastic Monolayer Cell Culture Flasks .4.Human Tumor Cells in Cuhure . . . . . .5.Evolution of a Cell Line.. . . . . . . . . . . .6. Structure of an Antibody Molecule . . .7. Preparation of Mouse Hybridomas and8. The Structure of DNA . . . . . . . . . . . . .9. The Process of Gene Expression . . . .

10. Recombinant DNA: The Technique ofSpecies With Those From Another.. .

Monoclinal Antibodies

Recombining Genes. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . 32

. . . . . . . . . . . . 40

Page

From One..,,. . . . . . . . . . . .

33353637394142

43

Chapter 3

The Technologies

Progress in the scientific techniques of biotech-nology clearly has affected society on manylevels–medical, social, economic, legal, and ethi-cal. Most of the technologies used to transformundeveloped human tissues and cells mentionedin this report can be categorized into three broadareas: tissue and cell culture technology, hybri-doma technology, and recombinant DNA technol-ogy. Advances in these technologies have in-creased our capability to identify and produceimportant human therapeutic agents. These fun-damental scientific techniques are having pro-found, practical impacts on our society. Thus, itis important to understand the nature of the basic

techniques and how they can be used to manipu-late tissues and cells into useful products in or-der to appreciate the novel legal, economic, andethical issues raised in this report.

The following brief review outlines the principaltenets of the three main techniques; the large-scalecommercial applications of these technologies arediscussed in another OTA report (23). While eachtechnology is reviewed individually, keep in mindthat it is the marriage of technologies that is thenorm—no single technology is the central elementin the development or commercialization of hu-man biological material.

TISSUE AND CELL CULTURE TECHNOLOGY

Cells are the basic unit of all living organisms.They are the smallest components of plants andanimals that are capable of carrying on all essen-tial life processes, A single cell is a complex collec-tion of molecules with many different activitiesall integrated to forma functional, self-assembling,self-regulating entity. Higher organisms and plantsare multicellular, with certain cells performingspecialized (i.e., differentiated) functions,

There are two broad classes of cells: prokaryoticand eukaryotic. The classes are basically definedby the manner in which the genetic material ishoused. Prokaryotes, generally considered thesimpler of the two classes, include bacteria. Theirgenetic material is not housed in a separate struc-ture (called a nucleus), and the majority ofprokaryotic organisms are unicellular. Eukaryotes,on the other hand, are usually multicellular organ-isms. They contain their genetic material withina nucleus, and have other specialized structureswithin their cell confines to coordinate differentcellular functions. The genetic material of eu-karyotic organisms is a structure called a chromo-some—a DNA and protein complex that is usuallyvisible to the eve with standard light microscopy.Humans are eukaryotes. Table 3 compares someof the features that distinguish microbial cells

Table 3.-Comparison of Microbial andMammalian Cells

Mammalian cellsCharacteristic Microbial cells (in culture)

Size (diameter) 1 to 10 microns 10 to 100 micronsMetabolic

regulation ... Internal Internal and hormonalNutritional

spectrum Wide range of Fastidioussubstrates

Doubling time Typically 0.4 to 2.0 hours Typically 12 to 60 hoursE n v i r o n m e n t Wide range of tolerance Narrow range of tol-

eranceSOURCE: Office of Technology Assessment, 1987

(prokaryotes) from cultured mammalian cells (eu-karyotes).

Multicellular eukaryotes are complex and diffi-cult, if not impossible, to examine in vivo at theorganismal level. Thus, scientists at the turn ofthe century began studying these organisms usinga reductionist approach, They dissected the manybiological processes in vitro by examining cellsisolated and maintained independently of a wholeorganism. This approach, called tissue and cellculture, has been refined considerably over theyears and the following section discusses this tech-nology as it applies to human cells. A separate sec-

31

— — —

32 . Ownership of Human Tissues and Cells

tion is devoted to a special application of cell cul-ture technology-making hybridomas.

Culturing Human Cells

The first experiments using tissue and cell cul-ture technology were conducted in 1907 whena scientist successfully grew frog nerve cells inculture (7). The technology was originally consid-ered a “model system” —a way for scientists toexamine physiological events outside an intactorganism. The approach was initially criticizedas myopic and artifactual, but tissue and cell cul-ture are now seen as fundamental scientific tools.These techniques are no longer only used as modelsystems, but are widely exploited techniques usedin biomedical research.

As a practical matter, the distinction betweentissue culture and cell culture is often blurred sothe terms are frequently used interchangeably.Strictly speaking, in cell culture technology sam-ples are removed from an organism and in vitromanipulation has destroyed the original integrityof the sample. In time, a sample isolated and estab-lished in the laboratory maybe called a cell line,In tissue culture, isolated pieces of tissue are main-tained with their various cell types arranged muchas they existed in the whole organism and theirfunctions remain largely intact, Tissue culturespresumably have more of their native identity,but are much more difficult to maintain than cellcultures.

Although many advances have occurredsince 1907, establishing a human cell culturedirectly from human tissue--called a primaryceil culture--is still a relatively difficult enter-prise. The probability of establishing a cell linefrom a given sample is low. Success can be un-dermined by contamination during collection andstorage, and is also dependent on how much dam-age the tissue suffered during collection of thesample. The success rate also depends on the typeof human tissue being used. Some cells are easyto culture—human skin fibroblasts and humanglial cells can be successfully established nearly100 percent of the times attempted (14,19). Others,however can be very difficult to establish. Somehuman tumors can be cultured with about a 10percent success rate (13).

While it is significantly less difficult to cultivatehuman cell lines than it is to establish them, work-ing with human materials is still much more prob-lematic than working with simpler organisms suchas bacteria or yeast. Nevertheless, scientists arecontinuing to make progress in developing optimalgrowth conditions and cell culture equipment.

The food required to sustain human cells in cul-ture is a liquid called growth medium. Differenttypes of human cells require different growth me-dia. Growth media are complex, and until recentlyanimal serum-containing many unidentified, butvital components —was a necessary ingredient ofall media. However, media with the identity andquantities of all components defined have beensuccessful in sustaining long-term growth of hu-man cells (8)20).

In addition to the many nutrient requirementsof human cells in culture, strict temperature con-ditions must be maintained. Variation in temper-ature exceeding 20 C from the optimum usuallyis not tolerated; higher temperatures in particu-lar are quickly lethal. Buffers are added to growthmedia to prevent drastic shifts in acidity, and themedia must be sterilized. Contamination of sam-ples during the early stages of culturing is a par-ticular concern, and rigorous care must be takento keep the culture free of contaminants such asyeast, fungi, bacteria, and viruses. Antibiotics andfungicides may be added to further discourageinfestation. Table 4 lists some of the requirementsfor successful cultivation of human cells in thelaboratory.

Table 4.—Some Nutrient and Growth ConditionRequirements for Culturing Human Cells

WaterSaltsSugarsVitaminsAmino acidsHormonesFatsBuffers (to maintain proper pH—i.e., prevent drastic shifts

in acidity)Gases (oxygen, nitrogen, carbon dioxide)Temperature (usually 98.6° F [37° C] for optimal growth)SterilizationAntibiotics and fungicides (optional)SOURCE: Office of Technology Assessment, 1987.

Ch. 3—The Technologies ● 3 3

Figure 3.—Plastic Monolayer Cell Culture Flasks

/ .

Photo credit: Ventrex Laboratories, Inc.

Cultured human cells grow as a suspension insolution, or attached to specially treated glass orplastic and submerged in growth medium (figure3). Human cells typically double in number in 18to 36 hours, compared to approximately 20 min-utes for the bacterium Escherichia coli. Samplesof human cells can be stored frozen in liquid ni-trogen ( – 1960 F) for future use. Certain typesof cells are more fragile than others, but with mod-ern freezing techniques most samples can bethawed and recovered decades later—often witha greater than 95 percent survivor rate.

Primary cell cultures are derived directly fromsolid human tissue or blood. In the case of cul-tures isolated from solid samples, extensive minc-ing or enzyme treatment maybe necessary to dis-perse the tissue. Since the earliest days of tis-sue and cell culture, it has been clear that notall the cells that are isolated from tissue andput into culture will survive. Thus, as soon asa sample is cultured it may not be representa-tive of the total specimen used, and the longerthe sample is in culture, the less it is like the

original specimen (2,4). For some liver cells, thefraction of cells resulting in viable outgrowth forany given sample is between only 1/1,000 to1/100)000 (0.01 to 0.10 percent) (10).

Primary human cell cultures typically maintainthe normal diploid number of human chromo-somes—46. They may also exhibit the functionsand properties indicative of their differentiatedorigin: liver cultures may produce certain liver-specific proteins or white blood cell cultures mayexpress their own specialized characteristics.

Cell cultures isolated from nontumor tissue havea finite lifespan in vitro (i.e., most cultures die af-ter a limited number of population doubling. )These cultures will almost always age unlesspushed into immortality by outside interventioninvolving viruses or chemicals. This aging phe-nomenon, called senescence, does not occur enmasse, but is a gradual deterioration and deathof the cell population. The type of tissue involvedand culture conditions are important variables indetermining cell lifespan. However, the age of thehuman tissue source is also a component, and thusprimary cell cultures can be studied as modelsof human aging.

Human Cell Lines

Long-term adaptation and growth of human tis-sues and cells in culture is difficult—often con-sidered an art—but it has been accomplished andmany established human cell lines (cells capableof continuous and indefinite growth in culture)exist. A primary culture that has been transformedinto an immortal cell line usually has undergonea “crisis” period. Most established cell cultureshave been derived from malignant tissue samples(figure 4). It is important to point out, however,that immortalization does not occur in all sam-ples isolated from tumors. As was mentionedearlier, certain types of tumors seem more likelyto establish continuous cultures. Figure 5 illus-trates the evolution of cultured cells.

It is not known precisely why a given samplegives rise to a continuous cell culture. It is possi-ble that a small number of cells in the originalsample become the immortal cell line. On the other

34 ● Ownership of Human Tissues and Cells

hand, one or a few cells may undergo a transfor-mation event during the “crisis” period to give riseto the immortal cell line. Evidence indicates thatthe latter explanation is more probable, but thepossibility that there is a subpopulation of the origi-nal sample with a predisposition to undergo thetransformation event cannot be discounted (4).

Established cell lines are usually aneuploid,which means that the number of chromosomesdeviates from the normal number of 46 for hu-mans. The first human tumor cell line, HeLa, wasisolated in 1951 (5). Derived from a cervical carci-noma, this widely used cell line has a chromosomenumber that varies from about 50 to 80, depend-ing on the particular isolate.

In addition to having aberrant numbers of chro-mosomes, established cell lines may not displaydifferentiated functions. Both of these properties

may be a result of the nature of the tumor usedto establish the cell line, or they may be the re-sult of changes the cells have undergone in orderto achieve continuous, long-term culture. Afterinitial immortalization, established lines are usu-ally isolated and expanded from a single cell—aprocess referred to as cloning. This means thatthe entire population of cells has resulted aftercontinual growth starting from a single cell.

Cells that have adapted to continuous culturecan not be considered entirely representative ofthe total population of the original isolate and theymay continue to change with time (4). Cloning isperformed, therefore, to provide a uniform pop-ulation of cells so that uniformity and accuracyin experimental results can be improved. But, con-tinuous growth of cells is a dynamic process—subpopulations of cells may suddenly acceleratetheir growth rate, shut down production of or


Figure 4.— Human Tumor Cells in Culture

Photo credit: Robyn Nlshimi

begin to overproduce compounds, or alter theirchromosome number. So in order to reproduceearlier experimental results, repeated subcloningof cultured cells may be required.

Using Cell Cultures

The applications of tissue and cell culture tech-nology are wide and varied. At both the basic re-

search and commercial levels, cell cultures areused as tools to study basic biological processes.A cell line may be used as a biological factory toproduce small or large quantities of a substance.Human proteins may be isolated directly from cul-tured cells. Cell cultures can also be the sourceof the genetic material needed to apply recombi-nant DNA technology in further studying a prob-lem. As will be described later in this chapter, cul-tured human cells, both primary and established,play an important role in recombinant DNA tech-nology. And finally, companies may use primaryand established cultures to test drugs or the tox-icity of compounds. The ability to maintain andmanipulate many types of human cell lines in acontrolled environment has expanded our knowl-edge of the biological sciences significantly andfacilitated biomedical research.

In addition to increasing our knowledge, nearly50 years after frog nerve cells were first culturedin vitro an important offshoot of growing cellsin culture was invented: a technique to fuse cellsfrom different sources. This technique, called cellfusion, has elucidated much of what is currentlyknown about:

● the structure and function of the humangenome,

● the expression and mechanism of heritableconditions,

● the regulation of normal biological reactions,and

● the processes of carcinogenesis and manyother diseases.

Cell fusion was also central to the developmentof hybridoma technology.

HYBRIDOMA TECHNOLOGY

Refinements in cell fusion (also called cell The immune response in higher animals serveshybridization) are responsible for the explosion to protect the organism against invasion and per-in hybridoma technology. Hybridomas are spe- sistence of foreign substances. It occurs only incial types of hybrid cells and to understand how vertebrates and is a cooperative effort among sev-they were invented and why they are important eral types of cells that results in a complex seriesit is helpful to understand the immune system. of events involving the production of antibodies

36 ● Ownership of Human Tissues and cc//s

Figure 5.— Evolution of a Cell Line

2C

18

16

14

12

10

8

6

u d 4 5 8 10 12 14 16

Weeks in culture

The vertical axis represents total cell growth on a log scale and the horizontal axis the number of weeks the hypothetical sam.ple has been in culture since it has been obtained from a donor. In this example, a continuous cell line is depicted as arisingat about 12.5 weeks. Different cultures will give rise to a continuous cell line at different times. In addition, senescence mayoccur in a sample at any time, but for human diploid fibroblasts it usually happens between 30 and 60 population doublings(10 to 20 weeks).

SOURCE: Adapted from R.1. Freshney, Culture of Anirna/ Cc//s: A Manua/ of Basic Technique (New York: Alan R. Liss, Inc., 1983),


and a class of molecules called lymphokines. Anti-bodies bind to a foreign invader, while lympho-kines are necessary for coordinating, enhancing,and amplifying an immune response. Both anti-body and lymphokine production operating inconcert are necessary for a complete and effica-cious response to a foreign challenge.

Scientists realized that obtaining a constant anduniform source of a single type of antibody wouldbe essential to understanding the intricacies ofthe immune response and that such a uniformsource of antibodies could provide a powerful,general analytical tool. High concentrations ofreliable antibodies and lymphokines also prom-ise rewards in diagnosing and treating human ills,The following two sections describe recently de-veloped technologies that yield pure antibodiesand higher concentrations of many lymphokines.

Monoclinal Antibodies

An antibody is a protein molecule with a uniquestructural organization that enables it to bind toa specific foreign substance, called an antigen.Antibody molecules have binding sites that arespecific for and complementary to the structuralfeatures of the antigen that stimulated their forma-tion. Antibodies formed by a sheep, for example,in response to injection of human hemoglobin (theantigen) will combine with human hemoglobin andnot an unrelated protein such as human growthhormone.

All antibodies are comprised of four proteinchains—two identical light chains and two identi-cal heavy chains. These subunits are always linkedin a fixed and precise orientation, as illustratedin figure 6. One end of the antibody contains twovariable regions, the sites of the molecule thatrecognize and bind with the specific antigen, Toaccommodate the many antigens that exist, thevariable end of an antibody differs greatly frommolecule to molecule. The other end of the anti-body is nearly identical among all structures andis known as the constant, or effecter, region. Theconstant region is not responsible for antibodybinding specificity, but has other functions.

other important actors in the immune responseare specialized white blood cells called lympho-cytes that are present in the spleen, lymph nodes,

Figure 6.—Structure of an Antibody Molecule

Constantregion

!

SOURCE: Office of Technology Assessment, 1984

and blood. A particular subclass of lymphocytes,called B lymphocytes or B cells, recognizes anti-gens as foreign substances and responds by pro-ducing antibodies highly specific for a given anti-gen. Any single B lymphocyte is capable ofrecognizing and responding to only one antigen.Once a B cell has been activated by an antigenit is committed to producing antibodies that bindto only that one specific antigen.

During an immune response to an invasion bya foreign substance (e.g., a virus), one of the eventsthat occurs within an organism is that many differ-ent B cells react and produce antibodies. Differ-ent B cells produce antibodies recognizing differ-ent parts (called determinants) of the virus, butas mentioned above, an individual B lymphocyteand its progeny produce only one specific kindof antibody. This multiple B cell reaction producesa mixed bag of antibodies with each type of anti-body represented in only limited quantities, andis called a polyclonal response. Polyclonal antibod-ies can be isolated from blood serum, and, untilrecently, were the principle source of antibodiesused by physicians and researchers. While anti-bodies produced this way were and still are use-ful tools to scientists and clinicians, a method to

38 . Ownership of Human Tissues and Cells

produce a constant and pure source of a singletype of antibody was still sought.

The discovery in 1975 of the technique to pro-duce a special hybrid cell known as a hybridomathat produces a specific type of antibody was, inthe words of one of the inventors, a “lucky cir-cumstance)” but one with profound effects forbiomedical research and commerce. Cesar Milsteinand Georges Kohler,l working at the Medical Re-search Council’s Laboratory of Molecular Biologyin Cambridge, England, used the well-establishedtissue culture technique of cell fusion to producea new type of hybrid cell—a hybridoma-capableof indefinitely proliferating and secreting largeamounts of one specific antibody (11,12).

Hybridomas are hybrid cells resulting from thefusion of a type of tumor cell called a myelomawith a B lymphocyte freshly isolated from anorganism (usually from the spleen or lymph nodes)that had been recently injected with the foreignsubstance of interest. Due to the recent exposureto the antigen, many of the B cells in such an organ-ism will be producing antibodies specifically com-plementary to the foreign substance just injected.This enrichment process is a key step in hybri-doma technology, since a human, for instance, iscapable of producing up to a million differentkinds of antibodies.

The hybridoma that results from the fusion ofthese two types of cells has characteristics of bothcells. As is often the case with tumor cells, themyeloma parent cell has the ability to grow andmultiply continuously in culture—it contributesthis characteristic of “immortality” to the hybri-doma. From the B cell, which is incapable of sus-tained growth and cultivation in vitro, comes theability to secrete a single, specific type of antibody.Thus, a particular hybridoma clone is a distinctcell line capable of continuously producing oneand only one kind of antibody—hence the namemonoclinal antibody. The culture conditions andtechniques used for hybridomas essentially arethose described for tissue and cell culture.

‘In this case, the antibody recognized a particular part of a sheepred blood cell. It is interesting to note that Milstein and Kohler didnot apply for a patent on this technique.

Independently isolated lines of hybridomas, eachoriginating from a single B cell fusing with a sin-gle myeloma cell, produce distinctive monoclinalantibodies. Each line is unique to the original con-tribution of the particular B cell parent. In thecase of Milstein and Kohler each different hybri-doma cell line isolated is an immortal antibody-producing factory targeted toward a different partof a sheep red blood cell. The method used to pro-duce mouse monoclinal antibodies is illustratedin figure 7.

Virtually all of the monoclinal antibodies cur-rently being used in humans as therapeutic agentsor imaging tools are rodent antibodies becausethe production of human hybridomas has beenmuch more difficult than the production of rodenthybridomas. To avoid some of the complicationsin patients treated with rodent antibodies, refine-ments in human monoclinal antibody technologywill be necessary. Researchers have developed in-genious in vitro methods and successfully isolatedsuitable immortal parental cell lines, so produc-tion of human hybridomas is rapidly progressing(16,17). Recently, researchers have developed apromising new method to produce large quanti-ties of human monoclinal antibodies (1).

The availability of large supplies of monoclinalantibodies is revolutionizing basic research, medi-cine, and commerce. Researchers have come tovalue monoclinal antibodies as important toolsfor dissecting the molecular structure and mech-anisms of genes; more often than not, monoclinalantibody technology is combined with recombi-nant DNA technology. High-volume productionof rodent monoclinal antibodies has had a signif-icant impact on the diagnostic industry in particu-lar. Monoclinal antibodies are reagents that areeasily standardized and provide reproducible re-sults, These substances have been adapted to clin-ical and home test kits, such as pregnancy diag-nostic kits, with much success. Use of monoclinalantibodies for prophylactic or therapeutic regi-mens in humans is in an embryonic stage.

Lymphokines

Two other specialized cell types involved in theimmune response are T lymphocytes and macro-phages. Like B lymphocytes, both of these cell

—

Ch. 3—The Technologies 39

Figure 7.— Preparation of Mouse Hybridomas and Monoclinal Antibodies

% Mouse isimmunized witha foreignsubstance or“antigen”

removed andminced torelease antibodyproducing cells(B lymphocytes)

Mouse spleen cells

Myeloma cellsare mixed andfused withB lymphocytes

i

SOURCE: Office of Technology Assessment, 1987.

types can detect and respond to the presence offoreign substances. However, rather than produc-ing antibodies, T cells and macrophages producea variety of protein molecules that regulate theimmune response. These molecules serve as mes-sengers that transmit signals between cells to or-chestrate a complete and efficient immune re-sponse against a foreign invader. The term“lymphokines” was coined in 1969 to describe thisgroup of nonantibody immune response modu-lators (3). Since that time, more than 90 lym-phokine activities have been described..

The products of thisfusion are grown in aselective medium. Onlythose fusion productswhich are both “immor-tal” and contain genesfrom the antibody-pro-ducing cells survive.These are called“hybridomas.”

Hybridomas are clonedand the resulting cellsare screened for anti-body production. Thosefew cells that producethe antibodies beingsought are grown inlarge quantities forproduction of mono-clonal antibodies.

Lymphokines may recruit other cells to partici-pate in and augment an immune response. Somelymphokines stimulate B cells to produce antibod-ies. Other molecules are released that suppressthe immune reaction or ensure that the systemfocuses on the irritant and does not run rampantin a nonspecific attack that would damage hosttissue.

Lymphokines are present in human blood in ex-tremely small amounts-on the order of parts perbillion. Interferon, for example, has been the most

———


widely examined Iymphokine to date by virtue ofits relatively “high” abundance. It takes approxi-mately 65,000 liters of blood to produce 100 mil-ligrams of interferon (21). A comparable taskwould be the search for less than one-eighth ofa teaspoon of salt in a swimming pool. Thus, sci-entists knew that to use lymphokines to treat hu-man illness would require a source yielding a high-quality, high-quantity sample.

In addition to the problem of obtaining suffi-cient quantities of these important biological reg-ulators, different lymphokines with antagonisticfunctions are often difficult to separate. In thepast, such impure preparations of lymphokineshave hampered efforts to understand the basicmechanism of how the immune system respondsto cancer or an agent of disease. Autoimmune dis-eases, for instance, are aberrations of the immunesystem resulting in an organism attacking itselfas a foreign substance. The availability of a lym-phokine drug to suppress an individual’s immuneresponse could alleviate much suffering. Similarly,other lymphokines could be used as therapeuticagents to boost a patient’s own immune systemto combat a foreign invasion.

Recent progress in obtaining pure lymphokinepreparations is a result of advances in cell cul-ture, hybridoma, and recombinant DNA technol-ogy. Scientists have now developed cell cultureconditions capable of sustaining continuousgrowth of cell lines producing elevated levels ofone or more lymphokines. Some of these lympho-kine-producing cell lines are derived from tumorcells that have been adapted to tissue culture con-ditions. Other cell lines have been isolated fromnormal cells that have been manipulated in a man-ner to transform them into immortal lymphokine-producing cultures.

The explosion in hybridoma technology also hasinfluenced the study and development of hybridT cell lines to produce lymphokines (6). Investiga-tors have had some success producing these hy-brid lymphokine factories, often referred to asT cell hybridomas. T cell hybridomas are the prod-

ucts of fusion events between immortal cancercells and isolated T lymphocytes.

Even though researchers have isolated and iden-tified many types of human cells producing lym-phokines, these cell lines are still not capable ofgenerating sufficient quantities of these moleculesfor widespread use. The human cell lines are veryimportant, however, as rich deposits of sourcematerial to clone lymphokine genes. Several dif-ferent genes have been cloned from human cellsthat produce measurable amounts of lymphokines(16), and once a particular lymphokine gene hasbeen cloned, large quantities of the protein mole-cule can be obtained via the methods developedfor large-scale production of recombinant DNAproducts.

Large-scale production of pure lymphokinesnow enables scientists to examine many aspectsof the immune system puzzle by manipulating cellsand lymphokines in vitro. The availability of com-mercial quantities of these pure immune regula-tors also affords physicians an opportunity to uselymphokines for treating human disease. Humanalpha-interferon has been approved by the Foodand Drug Administration (FDA) to treat certainmedical conditions and interleukin-2 is being usedin clinical trials to combat certain types of cancersor viral infections. Table 5 lists some of the lym-phokines that have been characterized and havereceived considerable attention for their possibleuse as human therapeutic agents.

Table 5.—Some Lymphokines WithTherapeutic Potential

InterferonInterleukin-l (also known as lymphocyte activation factor)lnterleukin-2 (also known as T cell growth factor)lnterleukin-3lnterleukin-4Colony stimulating factorsB-cell growth factorMicrophage activity factorT-cell replacing factorMigration inhibition factorSOURCE: Adapted from A. Mizrahl, “Biological From Animal Cells In Culture, ”

Biotechnology 4:123-127, 1966.


RECOMBINANT DNA TECHNOLOGY

History

In 1865, Mendel postulated that discrete biologi-cal units were responsible for maintaining char-acteristics in organisms from one generation tothe next. The faithful transmission, or inheritance,of these units—called genes—is common to theentire spectrum of living organisms, It is a resultof the remarkable capacity of a living cell to en-code, translate, and reproduce a chemical into itsultimate biological fate. The chemical responsi-ble for inherited characteristics is deoxyribo-nucleic acid, or DNA.

In 1965, a century after Mendel described theconcept and principles of inheritance, also calledgenetics, the term “genetic engineering” wascoined (9). The term genetic engineering is nowalso popularly referred to as “gene cloning” or“recombinant DNA .“ These techniques usually in-volve direct manipulation of the genetic material—the DNA-of a cell. Rather than rely on the appear-ance of spontaneous mutants or laborious extrac-tion of minute quantities of a valuable substancefrom tissue, it is now possible to use these tech-niques to isolate, examine, and develop a widerange of biological compounds quickly. Like theuse of cell culture, the use of recombinant DNAtechniques is a reductionist approach that has shedfurther light on the molecular details of regula-tion of many important biological processes, in-cluding arthritis, cancer, and development, Theprincipal advantages of these techniques are speedand ease of application.

Gene Cloning

DNA, which takes the structural form of a dou-ble-stranded helix (figure 8), is the informationsystem of living organisms. DNA in all organismsis composed, in part, of four chemical subunitscalled bases. These four bases—guanine (G), ade-nine (A), thymine (T), and cytosine (C)—are thecoding units of genetic information. These basesnormally pair predictably—A with G, and T withC—to form the DNA double helix structure. It isthe unique ordering of these bases in the helixthat determines the function of a given gene, and

Figure 8.— The Structure of DNA

SOURCE” Office of Technology Assessment, 1984

the complete blueprint for an organism is codedwithin its DNA.

There are two broad categories of genes: struc-tural and regulatory. Structural genes code forproducts, such as enzymes—proteins that cata-lyze biological reactions. Regulatory genes func-tion like traffic signals, directing when or howmuch of a substance is produced. The processwhereby the code of DNA is interpreted and aprotein synthesized is summarized in figure 9.

All cells, except egg and sperm cells and somecells of the immune system, contain the total in-formation capacity of the organism. Thus, the DNApresent in one human cell is identical to all othercells within the individual and has the capabilityof directing all possible functions. In individualhuman cells, however, not all functions operatesimultaneously.

The amount of DNA present in each cell of a

human being is 3.3 billion base pairs (15). About50,000 genes make up the complete human masterplan, and the average gene contains about 1,000


Figure 9.-The Process of Gene Expression

DNA

I

I Transcription

.

DNA

mRNA released andtransported to protein-synthesizing machinery

I Translation

+

Protein

During gene expression, the genetic material of an organismis decoded and processed into the final gene product (usuallya protein). In the first step, called transcription, the DNA dou-ble helix unwinds in the area near the gene, and a productcalled messenger ribonucleic acid (mRNA) is synthesized.This piece of mRNA is a single-stranded, linear sequence ofnucleotide bases chemically very similar to DNA and it iscomplementary to the section of the unzipped DNA. The sec-ond step of the process is called translation. The mRNA isreleased from the DNA, becomes associated with the protein-synthesizing machinery of the cell, and is decoded and “trans-Iated” into a protein product.

SOURCE: Office of Technology Assessment, 1987.

base pairs. Since this accounts for about only 50million base pairs, it is apparent that not all ofthe DNA within a human cell is devoted to modu-lating or specifying a particular gene product. Todate, specific functions have only been assigned toabout 50 million of the 3.3 billion base pairs pres-ent in humans. There is speculation that some ofthe unassigned 3.25 billion base pairs may containsome genes, but that much of the “excess” DNAis for architectural or other unknown functions.

Gene cloning refers to a process that uses a va-riety of procedures to produce multiple copiesof a particular piece of DNA. Since the amountof DNA in a human cell is enormous comparedto the amount present in an individual gene, thesearch for any single gene within a cell is likesearching for a needle in a haystack. Therefore,a range of tools have been developed that allowinvestigators to both identify a gene and amplifythe number of copies of the gene. As a metal de-tector allows easier detection of a needle in a hay-stack, and a photocopy machine reproduces doc-uments, “recombinant DNA technology” is a groupof methods that accelerates the investigation orproduction of genes. The specific details of thesemethods to join segments of DNA—sometimesfrom different species —vary from project to proj-ect and purpose to purpose. In general, however,all recombinant DNA methods require the fol-lowing:

●

●

●

●

a suitable vector,an appropriate host,a system to select host cells that have receivedrecombinant DNA, anda probe to detect the particular recombinantorganism of interest.

Perhaps the hallmark discovery that allowed sci-entists to clone genes was the isolation of natu-rally occurring enzymes in bacteria that recog-nize and cut DNA at specific strings of bases. Thestring of bases recognized by the enzyme is usu-ally four to six bases in length and depends onthe particular bacteria from which the enzymeis isolated. These enzymes, called restriction en-donucleases, are used in gene cloning to fragmentDNA into discrete, precise segments. Recentreports have described modified restriction en-

Ch. 3—The Technologies . 43

Figure 10.— Recombinant DNA: The Technique ofRecombining Genes From One Species With

Those From Another

enzvmes \New DNA

I

~ p r o d u c e s l a r g e p r o d u c e samount of DNA protein

Restriction enzymes recognize sequences along the DNA andcan chemically cut the DNA at those sites. This makes it pos-sible to remove selected genes from donor DNA moleculesto form the recombinant DNA. The recombinant molecule canthen be inserted into a host organism and large amounts ofthe cloned gene, the protein that is coded for by the DNA,or both, can be produced.

SOURCE: Office of Technology Assessment, 1987

zymes that are now capable of cutting DNA ata sequence of the investigator’s choice (18,22).With the aid of restriction enzymes, a particularfragment of DNA-often the gene of interest andsome neighboring bases-can be excised awayfrom large, unwieldy pieces of DNA.

Cloning human and other eukaryotic genes isusually more difficult technically than cloning bac-terial and viral genes. Refinements in recombi-nant DNA methods, however, have been invented.Figure 10 illustrates the basic technique for pre-paring a recombinant DNA molecule. The recom-binant molecule can be prepared in a number ofways, but ultimately the process involves linkingthe DNA sequence of interest to a second pieceof DNA known as the vector,

Vectors serve as vehicles for the isolation andhigh copy reproduction of a particular DNA frag-ment free from its normal environs. Vectors canbe bacterial, viral, phage, or eukaryotic DNA-orthey may be combinations of these DNAs. Thecharacteristics of vectors differ from construc-tion to construction. Some are capable of stablymaintaining a large piece of foreign DNA, somereproduce rapidly and in high copy number, whileothers, called shuttle vectors, can reproduce andfunction in both eukaryotic and prokaryotic cells.A critical consideration in commercial develop-ment of a cloned gene is the ability of the vectorto achieve high product expression.

The other principal player in a cloning systemis the host organism. Once foreign, or donor, DNAhas been inserted into the vector, the recombi-nant molecules must be introduced into an organ-ism that provides an optimal environment for in-creasing the number of copies of the cloned DNA,producing large amounts of a gene product, orboth. The host is often the bacterium Escherichiacoli, but human cells, yeasts, and other cells canbe suitable hosts. Mean generation time, ease ofculture, ability to stabilize and adjust to presenceof the vector(s), and ability to add sugar groupsto a gene product are some important factors toconsider in selecting a host.

In general, recombinant DNA technology worksin this sequence: first, donor DNA is cut by re-striction enzymes into many fragments, one ofwhich contains the sequence of interest. Thesedifferent fragments are joined with vector DNAto become recombinant DNA molecules. The re-combinant molecules are then introduced into thehost; for a variety of reasons, only some host cellswill take up the recombinant DNA. After this proc-ess, the fraction of host cells that received anyrecombinant DNA must be identified. This initialselection is often accomplished through the useof antibiotics that kill those host cells that did notreceive recombinant molecules.

Finally, the small number of recombinant organ-isms containing the specific donor DNA fragmentof interest must be found. This process is com-pleted via a tool that detects the gene or gene prod-uct of interest. This tool is called a gene probe.Examples of gene probes include a segment ofDNA similar to the gene of interest, but from a

—

44 ● Ownership of Human Tissues and Cc//s

different organism; a synthetic fragment of DNA been achieved, the host population containing thededuced from the protein sequence of a gene cloned gene can be expanded and the cloned geneproduct; a piece of RNA; or an antibody that binds used to identify, isolate, and scrutinize scarce bio-to the product of interest. logical compounds.

Once identification and purification of the ge-netically engineered (recombinant) organism has

SUMMARY AND CONCLUSIONS

Technologies grouped under the umbrella term“biotechnology” include tissue and cell culture,hybridoma technology, and recombinant DNAtechnology. Tissue and cell culture, the oldest ofthe three technologies, involves converting un-developed human biological materials into celllines capable of indefinite growth in a laboratory.Establishing human cultures is still a relatively dif-ficult enterprise, and the human cell line result-ing from any single sample has undergone manychanges. Continuous cultivation of cell culturesrequires stringent control of temperature, nutri-ent, pH, and sterile conditions. The use of humancell lines in research has contributed much to ourknowledge about human genetics and the regu-lation of normal and abnormal biological proc-esses. Cell lines also have been used for a broadrange of commercial purposes.

Hybridoma technology is a spinoff techniquefrom cell culture. Hybridomas are special hybridcells that are produced by fusing two types of cells:an antibody-producing B lymphocyte and a tumorcell called a myeloma. A hybridoma is capable ofmultiplying continuously in culture (a propertyit receives from the myeloma) as well as secret-ing antibodies with a single specificity (an abilitygained from the B lymphocyte). The antibodiesproduced by hybridomas are called monoclinalantibodies. Not only are monoclinal antibodiesimportant laboratory tools, but some are signifi-cant commercial commodities. one specific mousemonoclinal antibody was approved by the FDAin 1986 for use in the treatment of kidney trans-plant rejection.

Lymphokines are molecules that are secretedby specialized cells called T lymphocytes and mac-rophages. Many of these substances occur natu-rally in the human body, but were previously avail-

able in minute and usually impure amounts—ifat all. Lymphokines, also called bioregulators orbiological response modifiers, have significanttherapeutic promise in the treatment of a spec-trum of diseases because of their exquisite speci-ficity and reduced toxicity. Hybridoma, cell cul-ture, and recombinant DNA technologies permitlymphokines to be isolated in pure form and inquantities facilitating further analysis or use. Hu-man alpha interferon, a lymphokine produced bya combination of the biotechniques, was approvedin 1986 by the FDA for use in the treatment ofone form of leukemia.

Genes are composed of DNA and they are re-sponsible for the faithful inheritance of char-acteristics from one generation to the next. Re-combinant DNA technology, also called geneticengineering, involves techniques that allow directmanipulation of the genes—the DNA-of a cell.

Courtesy of: L. Gonich and M, Wheelis, The Cartoon Guide to Genetics


Gene cloning uses a variety of these recombinantDNA techniques to join segments of DNA, some-times from different species, in a form that al-lows multiple copies to be made. These multiplecopies can then be used to examine the regula-tion of a biological process, identify and isolatescarce compounds, or produce commercial quan-tities of an important substance. Three commer-cial products created through gene cloning—human growth hormone, human insulin, and hu-man alpha interferon –have been approved foruse in humans by the FDA.

CHAPTER 3

1. Casali, P., Inghirami, G., Nakamura, M., et al., “Hu-man Monoclonals From Antigen-Specific Selectionof B Lymphocytes and Transformation by EBV, ”Science 234:476-479, 1986.

2. Dendy, P.P. (cd.), Human Tumors in Short TermCulture: Techniques and Clinical Applications (NewYork: Academic Press, 1976).

3. Dumonde, D. C., Wolstencroft, R. A., Panayi, G. S.,et al., “ ‘Lymphokines’: Non-Antibody Mediators ofCellular Immunity Generated by Lymphocyte Ac-tivation, ” A’ature 224:38-42, 1969.

4. Freshney, R. I., Culture of Animal Cells: A Manualof Basic Technique (New York: Alan R. Liss, Inc.,1983).

5. (;eY, G.()., Coffman, W .D., and Kubicek, M. T., “Tis-

6

7.

sue Culture Studies of the Proliferative Capacityof Cervical Carcinoma and Normal Epitheliums,”Cancer Research 12:264, 1952.Ha”mmerling, G. J., Hammering, U., and Kearney,J. (eds.), Monoclinal Antibodies and T-cell Heybri-domas: Perspectives and Technical Advances (NewYork: Elsevier/North Holland, 1981).Harrison, R. G., “Observations on the Living Devel -oping Nerve Fiber, ” Proceedings of the So;iettiv forExperimental BiologV and Medicine 4:140-143,1907.

8. Hayashi, I., and Sate, G., “Replacement of Serumby Hormones Permits Growth of Cells in a Definedhledium,” Nature 259:132-134, 1976.

9. Hotchkiss, R. D., “Portents for a Genetic Engineer-ing, ” Journal of Heredi{v 56:197, 1965.

10. Kaighn, M. E., “Human Liver Cells,” Tissue Culture:Methods and Applications, P,F. Kruse, Jr., and M.K.Patterson, Jr. (eds. ) (New York: Academic Press,1973).

The ease of application of biotechnology proc-esses has allowed researchers to turn undevelopedhuman tissues and cells into human biologicalproducts with significant therapeutic promise andcommercial potential. Yet the ultimate value ofthese technologies may not be simply their endproducts; their greater value may be the insightsthey provide about disease processes.

REFERENCES

11. Kohler, G., and Milstein, C., “Continuous Culturesof Fused Cells Secreting Antibody of PredefineSpecificity, ” Nature 256:495, 1975.

12. Kohler, G., and Milstein, C., ‘(Derivation of SpecificAntibody-Producing Tissue Culture and TumorLines by Cell Fusion, ” European Journal of Immu-nology 6:511, 1976.

13. Lasfargues, E. Y., ‘(Human Mammary Tumors,” Tis -

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sue Culture: Methods and Applications, P.F. Kruse,Jr., and M.K. Patterson, Jr. (eds.) (New York: Aca-demic Press, 1973).Martin, G. M., “Human Skin Fibroblasts, ” Tissue Cul-ture: Methods and Applications, P.F. Kruse, Jr., andM.K. Patterson, Jr. (eds.) (New York: AcademicPress, 1973).McKusick, V. A., The Johns Hopkins UniversitySchool of Medicine, Baltimore, MD, personal com-munication, May 1986.Mizrahi, A., “Biological From Animal Cells in Cul-ture,” Bio/Technology 4:123-127, 1986.Pinsky, C. M., ‘(Monoclinal Antibodies: Progress IsSlow But Sure, ” New England Journal of Medicine315(11):704-705, 1986.Podhasjska, A.J., and Szybalski, W., “Conversionof the F’okI Endonuclease to a Universal Restric-tion Enzyme: Cleavage of Phage M13mp7 DNA atPredetermined Sites, ” Gene 40:175-182, 1985.Pont&, “Human Glial Cells, ” Tissue Culture: Meth-ods and Applications, P.F. Kruse, Jr., and M .K. Pat -terson, Jr. (eds.) (New York: Academic Press, 1973).Sate, G., “The Role of Serum in Cell Culture, ” Bio-chemical Action of Hormones, G. Witlack (cd. ) (NewYork: Academic Press, 1975).Stewart, W. E., The Interferon S&vstem (Wien, ATY:S~ringer-Verlag, 1981).

46 ● ownership of Human Tissues and cc//S

22. Szybalski, W., “Universal Restriction Endonucle- 23. U.S. Congress, Office of Technology Assessment,ases: Designing Novel Cleavage Specificities by Commercial Biotechnology: An International Anal-Combining Adapter Oligodeoxynucleotide and En- ysis, OTA-BA-218 (Elmsford, NY: Pergamon Press,zyme Moieties, ” Gene 40:169-173, 1985. Inc., January 1984).

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