cancer research · cancer research volume26 august 1966 number8 [cancer research 26 part 1,...

Cancer ResearchVOLUME26 AUGUST 1966 NUMBER8

[CANCER RESEARCH 26 Part 1, 1597-1605, August 1966]

Crown Gall Tumorigenesis

II. Relations between Wound Healing and the Tumorigenic Response1

JACQUES LIPETZ

Laboratory of Plant Morphogenesis, Manhattan College, Bronx, New York

Summary

Studies of the wound-healing process and the size of crowngall tumors formed in KalanchoÃ«plants at 25, 32, and 36Â°C

have demonstrated that: (a) the time required for the 1st celldivision as a result of wounding is linearly dependent upon temperature; (6) jilants maintained at higher temperatures reach apeak of sensitivity to the tumor-inducing principle more rapidlythan those plants maintained at lower temperatures; (c) theplateau of maximum sensitivity is temporally shorter at thehigher temperatures; (d) the wound-healing process and theprocess of conditioning can be correlated; (e) maximum hostsensitivity is achieved just prior to the 1st wound-induced celldivision; (/) the "transformation" process requires 8-10 hr, anda prior 8-10 hr are required for bacterial environmental adjustment. These data suggest that there exists a sensitive stage inthe host cells between quiescence and their 1st wound-induceddivision. If this period is of sufficient duration tumorigenesiswill occur provided the virulent bacteria are present and "environmentally adjusted."

Introduction

Crown gall was the 1st experimentally produced neoplasticdisease. As such it has served as an experimental model for studies in both plant and animal oncology (2). From studies on thisdisease the concept of sensitive or conditioned cells, i.e., thepresence of a stage in a host which renders it sensitive to a tu-morigenic agent, has been developed (1). This concept may explain the observations that in all plant and some animal tumors,trauma greatly increases the response to a tumorigenic stimulus(13, 16).

During the course of their response to a wound stimulus, cells

1Supported by Grant Ã‡AOC955from the National Cancer Institute, NIH. Some of the preliminary work included here wassupported by Postdoctoral Fellowship CF7607 from the samesource. The Laboratory of Plant Morphogenesis is supported inpart by USPHS Institutional Grant RC 1193, Damon RunyonFund Grant DRM 710, and the Christine Sonntag Foundation.

Received for publication November 29, 1965.

in the wound area of susceptible plants become sensitive to anas yet unisolated and uncharacterized agent, referred to as thetumor-inducing principle (TIP), elaborated by the soil microbe,Agrobacterium tumefaciens (Conn), which can "transform" thesecells into tumor cells. It has been repeatedly demonstrated thatthe TIP can only act upon cells responding to a wound stimulus(4). Such cells remain in a sensitive or "conditioned" state for a

limited period of time, then once more become refractory to thetumorigenic stimulus (1). In KalanchoÃ«conditioning can occurat both 25 and 32Â°C,although tumorigenesis only occurs attemperatures below 30Â°C(1). Recently I have demonstratedthat the conditioning process is accelerated at 32Â°C(15).

Cells exposed to the TIP at the peak of their conditioningcycle form large, rapidly growing tumors. In contrast cells exposed to the same stimulus prior to or after the peak of conditioning form small, slowly growing tumors (1).

A comparison of conditioning and wound healing at 25Â°CledBraun and Mandle (4) to suggest that the "transformation of anormal cell to a tumorous cell occurs... just before or duringthe earliest stages of active wound healing." This report will

examine the relationships between conditioning and woundhealing at 3 temperatures in order to more fully define the nature of the conditioned cell.

Materials and Methods

Virulent strain B6 of Agrobacterium tumefaciens (Smith andTown). Conn was originally obtained from Dr. Armin C. Braunof Rockefeller University and maintained on nutrient dextroseagar. Inocula of known cell number were prepared in the following manner. Bacteria were transferred from a 24-hr-old agarslant into defined liquid medium (19) and allowed to grow for24 hrs. An aliquot was then transferred to fresh medium andshaken on a rotary shaker. Bacterial number was determinedturbidometrically with a Coleman Jr. spectrophotometer. Optical density readings were converted into cells/ml by means ofa previously prepared calibration chart.

KalanchoÃ«daigremontiana (Hamet and Perrier) plants werevegetatively propagated from stock originally obtained from the

AUGUST 1966 1597

on May 28, 2020. © 1966 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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Jacques Lipetz

100

90

80o

E 70uEUCL~ 60

OD

i 50

It 40

o

i30

IO

o 32'Câ€¢I6hr 32'CÂ°9hr 32'C*3hr 32'C

IO 15 20 25 30 35 40 45 50

TIME AFTER WOUNDING (HOURS)

T- CHART 1. Effect of temperature on the formation of wound cambium. Plants wounded and maintained at 25Â°C(open triangles),32Â°C (open circles), 36Â°C (closed circles). Plants wounded andmaintained at 32Â°Cfor 3 hr (cloned triangles), 9 hr (open squares),and 1C hr (closed squares) prior to being placed at 25Â°C.The 1st

observable cell divisions occur between the 1st marked point oneach curve and the A"axis. Each point represents the average of at

least 15 wounds studied.

department of Plant Pathology at Rockefeller University. Plants10- to 12-cm high were wounded by means of a sterile lancetthrough the 3rd and 4th youngest internodes. After wounding,the plants were placed in growth chambers maintained at thedesired temperature. Temperature variations in the chambersnever exceeded Â±1.5Â°C,and relative humidity was maintainedat 70-80f;Â¿;light intensity at the bench top was approximately650 foot candles.

The level of conditioning in wounded plants was determinedas follows. Plants were wounded with a sterile needle and placedat the desired temperature. At the times stated in "Results"plants were inoculated with ~106 bacteria and placed at 25Â°Cfor 24 hr to allow "transformation" to occur. At the end of thistime the plants were placed at 32Â°Cto stop further "transformation" and kept there for 21 days. After this 3-week period

tumors were scored by comparing them to previously publishedphotographs, which represent a spectrum of increasing tumorsize ranging from 0 to 2 (15).

The minimum time required for tumor induction, and thetime required for bacterial environmental adjustment, were de

termined in a similar fashion, taking advantage of the ability tolimit the length of bacterial action by placing the infected plantat 32Â°C.Further experimental details are given in "Results."

Wound healing was studied by methods previously described,in which the degree of wound cambium formation is scored (15).Figs. 1-4 illustrate representative points of the scoring method.

Results

EFFECT OF TEMPERATURE ON WOUND HEALING. As Can be Seen

from the data in Charts 1 and 2, the time between woundingand the appearance of the 1st visible oriented cell divisions islinearly and inversely related to temperature in the range 21-36Â°C.The time (hr) at which such divisions are detectable TICD

in the wound healing process can be expressed as:

TICD =1.5 (temperature Â°C)+ 70

' Plants exposed to 32Â°Cfor short periods of time after wound

ing initiate oriented cell divisions sooner than plants maintainedcontinuously at 25Â°C.This reduction in time to achieve TICD is

also inversely and linearly related to temperature as shown inChart 3. This time difference, ATico, is related to length of exposure at 32Â°C,provided this exposure exceeds 3 hr and is no

longer than 16 hr, and can be expressed as follows:

ATicD = 0.5 (hr at 32Â°C)+ 3.5

EFFECT OF TEMPERATURE ON CONDITIONING. The Conditioning

curves shown in Chart 4 show that increasing temperaturesaffect the conditioning cycle in 2 major respects: the time required for conditioned cells to reach a level at which they canform tumors scored as 1.0 (Ti.0); the length of time this level ismaintained (Tp). Both of these variables are inversely and linearly related to temperature within the range of 25-32Â°C.These

data, obtained from Chart 4 and plotted in Chart 5, can be expressed as follows:

TÃo= â€”1.5(temperature of conditioning) +57.5

T,, = 1.6 (temperature of conditioning) + 74

The time required to reach a level of conditioning less than1.0 after the peak is also inversely related to temperature. Thisrelationship can be expressed as follows:

Time to end peak = -3 (temperature) + 126

COMPARISON OF WOUND HEALING AND CONDITIONING DATA.

The data presented in Chart 1 indicate that the 1st orientedcell divisions occur 36 hr after wounding in plants maintainedat 25Â°C.This period is shortened by 12 hr if the plants are maintained at 32Â°Cand by 18 hr in plants maintained at 36Â°C.Thus,at any given time after wounding, plants maintained at 36Â°Ccan

be considered as being 18 hr further along in the wound-healingprocess and plants maintained at 32Â°C12 hr further than thoseat 25Â°C.Thus 3 hr at 36 = 21 hr at 25Â°Cwhich equals 15 hr at32Â°C.Tables 1 and 2 compare the size of tumors formed bywounds conditioned at 25, 32, and 36Â°Cat corresponding periods

in the wound-healing cycle.The data presented in these tables show that from the 6th to

the 12th hr after wounding, plants conditioned at 25, 32, or36Â°Cform tumors of the same size if the comparison is made

1598 CANCER RESEARCH VOL. 26



Crown Gall Tumorigenesis. II

I

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LÃ¼

40

35

30

25

20

IS

IO

yâ€”l.5x+70

J I J I21 23 25 27 29 31 33 35 37 39

TEMPERATURE ("O

CHART2. Relation between temperature and the time of the 1st observable cell division. These data are derived from Chart 1.

12

IO

LUO

coCL

eouer

y*O.5x+3.5

I I I6 9 12TIME AT 32Â°C(HOURS)

15 18

CHART3. Reduction in time required for 1st observable cell divisions by pretreatment at 32Â°C.The doited line between 0 exposureto 32Â°C,that is continuous exposure to 25Â°C,and a 3-hr exposure to 32Â°Crepresents the possible kinetics of a "lag" phase. These data

are derived from Chart 1.

between equivalent times in the wound-healing process. Comparison of these 2 events at 25 and 32Â°Creveals a close corre

spondence from the 3rd to the 27th hr after wounding.Chart 4 demonstrates that plants conditioned at 36Â°Creach

the 1.0 level of conditioning 17 hr prior to plants conditionedat 25Â°Cand plants conditioned at 32Â°Creach this point 11 hrearlier than plants conditioned at 25Â°C.These time differences

are within 1 hr of the time differences between the 1st emergence of oriented cell divisions at these same 3 temperatures(Chart 1).

Chart 6 illustrates further relations between the conditioningcycle and the wound-healing process. These can be summarizedby the following equations:

Time to reach 1.0 level of conditioning = T1CD â€”15

Time required to reach end of 1.0 level of

conditioning = 2 T1Co â€”16

Tn = Tion ~2

AUGUST 1966 1599



Jacques Lipetz

1.75

1.50

1.25

te.8 1-00V)

0.75

0.50

0.25

0.00 l l l12 18 24 30 36 42 48 54 60

TIME AFTER WOUNDING (HOURS)CHART4. Relation between tumor score and length of time between wounding and infecÃion at 25Â°C(open circles), 32Â°C(open squares),

and at 36Â°C(open triangles). Plants were wounded, placed at the desired temperature for the number of hr indicated, then infected withbacteria, allowed to remain at 25Â°Cfor 24 hr, then placed at 32Â°Cto cut off further tumor induction.

Ul

55

50

45

40

35

30

25

20

15

IO

A Timeto reach IÃ›o Timeto end1.0Â°Lengthof peak

I

TABLE 1COMPARISONor TUMORSIZE AT EQUIVALENTTIMES AFTER

WOUNDINGAT25, 32, AND36Â°C

HRAFTERWOUNDINGAT36Â°C309121518CORRESPONDING

TIME(hr)AT25Â°C21242730333632Â°C151821242730TUMORSCORE25

Â°C1.001.401.451.451.401.4532Â°C1.501.501.501.451.300.8036Â°C0.801.501.501.351.100.90

23 25 27 35 3729 3l 33TEMPERATURE (Â°C)

CHART5. Relations between temperature and the time requiredto reach 1.0 tumor levels (open triangles), to end this level (opencircles) and the length of this plateau (open squares). Data derivedfrom Chart 4.

It can thus be seen that there are clear relationships betweenthe wound-healing process at various temperatures and conditioning at the same temperatures.

TIMEREQUIREDTOFORMTUMORS.Braun (1) has reported thatthe bacteria require a period of "environmental" adjustment inthe plant, which can occur at 32Â°C.After this bacterial environ

mental adjustment time, he reported that tumors could beformed if the infected plants were exposed to 25Â°Cfor 8-10 hr.

In order to measure the bacterial environmental adjustmenttime (B.E.A.T.), cells were conditioned at 32Â°Cfor 24 hr. Bac

teria were then introduced into the wound 14, 8, 6, 3, or 0 hrprior to the end of the 24 hr of conditioning. At the end of theconditioning at 32Â°C,the now infected plants were placed at25Â°Cfor periods of time ranging from 4 to 18 hr, to allow "transformation" to occur. At the end of each "transformation" periodthe plants were placed at 32Â°Cto prevent further "transformation." Two weeks later tumors were scored as previously de

scribed. An average score of 0.2 or higher was considered as evidence of "transformation."

KiOO CANCER RESEARCH VOL. 26




TABLE 2

COMPARISONOF TUMOR SIZE AT EQUIVALENTTIMES AFTERWOUNDINGAT 25 AND 32Â°C

55r

HR AFTER WOUNDINGAT32Â°C369121518212427CORRESPONDINGTIME(hr) AT25Â°C1518212427150333639TUMOR

SCORE25Â°C0.650.851.051.301.401.451.451.451.4532Â°C0.700.851.001.201.501.501.501.451.30

The data presented in Chart 7 demonstrate that in the presenceof conditioned cells the B.E.A.T. can be as short as 8 hr. IfB.E.A.T. is less than 8 hr the time of 25Â°Cexposure of the in

fected plant must be correspondingly increased. No tumors areformed if the sum of B.E.A.T. + exposure time at 25Â°Cis lessthan 16-18 hr, or if the time at 25Â°Cis less than 8-10 hr.

EFFECT OF INCREASED EXPOSURE OF CONDITIONED CELLS TOBACTERIA.If couditiod cells are exposed at 25Â°Cto the bac

teria for longer than the minimal 16 hr total exposure requiredto form tumors, the size of the ensuing tumor increases linearlywith the increased length of exposure. Although the size of thetumor formed varies with the length of time cells are conditioned,the rate of increase of tumor size appears to be independent ofconditioning and linearly dependent upon the length of timecells are exposed to the action of the bacteria (Chart 8). It canalso be noted from the data presented in Chart 8 that the totaltime of bacterial exposure required to form 0.2 tumor is increasedin the presence of cells conditioned for short periods of time.

Discussion

EFFECTOF TEMPERATUBEON WOUNDHEALING.The stimulation by wounding of normally quiescent plant and animal cellsinto mitotic activity has been known for some time. Although awound hormone has been isolated from plant tissue (11), its activity appears to be limited to the tissue from which it wasoriginally isolated. The control of mitotic activity in animalcells by mitosis-inhibiting substances or chalones has been re

viewed by Bullough (7, 8). In plants the observations that themitotic response in wounded plants can be decreased or evenprevented by washing the wound area shortly after wounding(14) sap from wounded tissue can stimulate normally quiescenttissue to division (21) and the plane of division of the woundcambium is for the most part parallel to the plane of the wound(17) suggest that wounded cells produce some mitotic stimulant.

Recently it has been demonstrated that a substance known as"retine" may play a role as an inhibitor of cell division; its action

is balanced by a cell division stimulator known as promine (20).Such a control mechanism has not as yet been demonstrated inplants; although retine has been isolated from plant cells itsactivity has only been assayed in animal systems (10).

90Timeto reach1.0

- o Timeto endl.On Length of peak

cote.

40

35

30

uI 25I-

2O

15-

10

x-15

I10 20 30 40 50

TIME TO FIRST CELL DIVISION(HOURS)

60

CHART 6. Relations between wound healing, conditioning, andtemperature. The time required to reach the 1st cell division at25, 32, and 36Â°Crelated to the time required to reach 1.0 tumor

score (open triangles), end 1.0 tumor score (open circles) and thelength of the peak (open squares). Data derived from Charts 1 and4.

The time interval between the actual physical wounding andthe 1st appearance of oriented cell divisions might thus representthe time required for wound hormones to reach effective levels,the time required for retine or other cell division inhibitor levelsto be lowered, the time required for promine or other cell divisionstimulators to reach an effective level, or a shift in retine/promineor inhibitor/stimulator ratio.

In addition to these 4 possibilities it must be borne in mindthat time may be required for cells to respond to any of the abovesituations.

Whatever the stimulatory mechanism, 3 aspects of its actionare now known in KalanchoÃ«: the time required for itsaction is inversely and linearly related to temperature between21 and 36Â°C;short exposures (3-16 hr) to higher temperatures

followed by a return to a lower temperature decrease the timerequired for action directly and linearly with temperature (according to the relation: reduction in time for 1st cell division =0.5 [time at 32Â°C]+ 35) ; once even a small fraction of the cell

population has begun to react to the stimulus, the rest of thepopulation does so at a constant rate which is independent oftemperature.

AUGUST 1966 1601



Jacques Lipetz

20

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OC Ã¯

16

o*CM

l2

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O 4-

l l l l l4 8 12 16 20 24

BACTERIAL ENVIRONMENTAL ADJUSTMENT TIME (HOURS)

CHART7. Time required for B.E.A.T. and for tumor induction in the presence of conditioned cells. Plants were wounded, placed at32Â°Cfor 24 hr, infected with bacteria and allowed to remain at 32Â°Cfor 2-24 hr, kept at 25Â°Cfor 6-18hr, then returned to 32Â°Cfor the

duration of the experiment. Vertical lines represent range, not S.D.

1.2

1.0

0.8Idg

K

Â§ 0.4

0.2

0.0

Â°/2t>r 32'CÂ°24hr 32'CA3fir 32'C

I I I I14 3016 18 20 22 24 26 28

TIME OF BACTERIAL CONTACT AT 25Â°C( HOURS)

CHART 8. Relation between tumor size and length of contact of cells with bacteria at 25Â°C.Plants were conditioned at 32Â°Cfor 12hr (open squares), 24 hr (open circles), or 3 hr (open triangles), infected with bacteria and placed at 25Â°Cfor 16, 20, 24, or 28 hr, thenreplaced at 32Â°Cto cut off further tumor induction.

The 4 alternate hypotheses on the mechanism of stimulationof cell division by wounding can be generalized to sufficientsynthesis and sufficient destruction. Using this generalizationthe data presented in Charts 1-3 may tentatively be interpretedas indicating that as a result of wounding certain processes, P,are initiated which lead to the synthesis of mitotic stimulants orthe destruction of inhibitors. This process is directly related totemperature. Thus short exposures to higher temperatures temporarily increase the rate of P. Since an exposure of less than 3hr to 32Â°Cdoes not appear to change the time required for the

1st cell divisions in the same way longer exposures do, this timeperiod (3 hr postwounding) can be considered as a "lag" periodprior to the activation of P. The presumed kinetics of this "lag"

period are indicated by the dotted line in Chart 3.Once the critical level of mitotic stimulant, or inhibitor, is

reached the cells begin their biochemical preparations for division. Since, at all the temperatures studied, the time betweenthe 1st observable cell divisions and the formation of a dividingwound cambium is the same; it is likely that this 12-hr periodrepresents some temperature-insensitive process. Alternatively,





it can be postulated that once a given level of mitotic stimulatoris reached its synthesis decreases or stops, or that the synthesisof a mitosis inhibitor increases. Thus the time required to synthesize the level of stimulator required to initiate mitosis canbe temperature dependent, but its ensuing action can be temperature independent.

EFFECTOFTEMPERATUREONCONDITIONING.The time requiredfor cells to reach a given level of conditioning, the time theyremain at that or a greater level, and the time at which the levelof conditioning begins to drop are all inversely and linearly related to temperature as shown in Charts 5 and 6. Furthermorethe length of time a conditioning plateau of 1.0 or higher is maintained is 15 hr longer than the time required to reach this plateau.This relationship can be expressed as follows:

Length of plateau = time to achieve plateau + 15

The availability of data on the effect of temperature on bothconditioning and wound healing now make it possible to comparethese 2 processes in the hope of further defining the conditionedcell.

RELATIONSHIPS BETWEEN WOUND HEALING AND CONDITIONING.

Braun (l) demonstrated that cells in the vicinity of a woundgradually become increasingly susceptible to the action of A.tumefadens. The events occurring in the host cells during thisperiod of time required to reach sensitivity are referred to asconditioning; cells susceptible to "transformation" are known as

conditioned cells. In the same paper Braun also demonstratedthat as the conditioning process progresses, larger tumors areformed. Some time after wounding, the cells in the wound areabecome less susceptible to "transformation" and eventually lose

their susceptibility to the tumorigenic stimulus. Conditionedcells can only be recognized by their ability to respond to thetumorigenic stimulus produced by A. tumefaciens. The abilityto identify these cells by other means would be of importance inthe understanding of the tumorigenic process.

Cells transformed early or late in the conditioning cycle formsmall tumors, whereas cells transformed at the peak of the cycleform large tumors. This difference in tumor size can be accountedfor in 2 ways: (a) More cells are available for transformation atthe peak of the cycle; thus more tumor cells are formed and theeventual size of the tumor is larger. (6) Cells transformed at thepeak of the cycle undergo a more malignant change leading tomore rapidly dividing cells. This latter possibility has beendemonstrated in crown gall tumors of Vinca (3).

The data presented in Chart 8 indicate that the rate of increase in the size of tumors in Kalanchoe is dependent on thelength of time the conditioned cells are exposed to the action ofthe bacteria. The ultimate size of the tumor is of course dependent upon the stage of conditioning at which the tumorigenicstimulus is given, as well as the length of time it is given. Thesedata suggest that the conditioning process involves the gradualformation of an increasing number of conditioned cells, the attainment of a maximum size of this pool, and its eventual gradualdecrease.

At all 3 temperatures studied 3 correlations between the conditioning process and the wound-healing process are clearlyevident, (a) The time required to reach a 1.0 level of conditioning, which can now be inteq^reted as the time required to reacha plateau in the number of conditioned cells, is 15 hr less than

the time required to initiate the 1st oriented cell divisions. Thisis consistent with the hypothesis suggested by Braun (4) thatcells are transformed prior to division, (o) The time required toreach a decrease in tumor size, which can now be interpreted asthe time required for the size of the pool of conditioned cells todecrease, is the same as the length of time required for woundsat 25Â°or at 32Â°Cto form a complete wound cambium. At 36Â°C

this decrease in the size of the pool occurs at the same time asthe 1st oriented cell divisions become observable. These datasuggest that the cells, temporarily between the quiescent stateprior to their response to a wound stimulus and their 1st divisionresulting from this stimulus, are susceptible to "transformation."

(c) If the difference in time required for the 1st oriented cell divisions to occur is taken into account, there is a close correspondence between the wound-healing process at the 3 temperatures studied and the conditioning process at these temperatures.These findings suggest that the size of the pool of conditionedcells at a given time reflects the stage in the wound-healing process prior to the 1st observable cell divisions.

Recent studies have demonstrated that cells go through 3distinct stages between mitoses. These stages are referred to asGÃ¬,S, and G2. These 3 stages and the mitotic process itself areknown to vary in length and to be independently affected bytemperature (5, 6, 12, 18). Since the data presented here demonstrate that a cell is conditioned prior to mitosis, it is reasonableto suggest that there is a sensitive stage between quiescence andmitosis during which the tumorigenic stimulus can be effective.The existence of such a sensitive stage has also been recentlysuggested for radiation carcinogenesis in mice (9). The time required to "transform" a conditioned cell to a tumorous cell is at

least 8 hr. Thus if the length of the sensitive stage is less than8 hr no tumors will be formed. This hypothesis might accountnot only for the failure of Kalanchoe to form tumors at elevatedtemperatures (4) but for the failure of certain cells, which dodivide as a result of wounding, to form tumors in the presenceof the bacteria. The technic now available for the measurementof the various premitotic stages should make it possible to learnwhich of these stages is sensitive to the tumorigenic stimulus.

Acknowledgments

I am grateful to my colleagues at the Laboratory of PlantMorphogenesis for many helpful discussions and particularly toDr. Robert E. Beardsley for his suggestions on the mathematicaltreatment of the data. Mrs. B. Del Bene was invaluable as a sourceof technical aid.

References

1. Braun, A. C. Conditioning of the Host Cell as a Factor in theTransformation Process in Crown Gall. Growth, 16: 05-74,1952.

2. . The Physiology of Plant Tumors. Ann. Rev. PlantPhysiol., 5: 133-62, 1954.

3. . A Phj'siological Basis for Autonomous Growth of theCrown-Gall Tumor Cell. Proc. Nati. Acad. Sci., 44: 344-49,1958.

4. Braun, A. C., and Mandle, R. J. Studies on the Inactivationof the Tumor-inducing Principle in Crown Gall. Growth, 12:255-69, 1948.

5. Brown, R. The Effects of Temperature on the Duration of the

AUGUST 1966 1603



Jacques Lipetz

Different Stages of Cell Division in the Root-Tip. J. Exptl.Botany, 2: 96-110, 1951.

6. Brown, II., and Rickless, P. A New Method for the Study ofCell Division and Cell Extension with Some PreliminaryObservations on the Effect of Temperature and of Nutrients.Proc. Roy. Soc. London, Ser. B, 136:110-25,1949.

7. Biillough, W. S. Growth Regulation by Tissue-specific Factors,or Chalones. In: P. Emmelot and 0. MÃ¼hlbock (eds.) CellularControl Mechanisms and Cancer, pp. 124-45. New York:

Eisevier, 19G4.8. â€” â€”. Mitotic and Functional Homeostasis. A Speculative

Review. Cancer Res., U: 1683-1727, 1965.9. Cole, L. J., and Nowell, P. C. Radiation Carcinogenesis; the

Sequence of Events. Science, 160: 1782-86, 1965.10. Eg.vÃ¼d,L. G., Studies on Autobiotics: Chemical Nature of

Retine. Proc. Nati. Acad. Sci. U.S., 04: 200-2, 1965.11. English, J., Jr., and J. Bonner. The Wound Hormones of

Plants. I. Traumatin, the Active Principle of the Bean Test.J. Biol. Chem., 121: 791-99, 1937.

12. EVANS,H. J., ANDSAVAGE,J. R. K. The Effect of Temperatureon Mitosis and on the Action of Colchicine in Root MeristemCells of Vicia, Jaba. Exptl. Cell Res., 18: 51-61, 1959.

13. Gottfriedâ€”B., Molomut, N., and Patti, J. Effect of Repeated

Surgical Trauma on Chemical Carcinogenesis. Cancer Res.,21:-658-60, 1961.

14. Haberlandt, G. Wundhormone als Erreger von ZellteilungenBeitr. Allgem. Botanik, 2: 1-54, 1921.

15. Lipetz, J. Crown-Gall Tumorigenesis: Effect of Temperatureon Wound Healing and Conditioning. Science, Â¡49:865-67,1965.

16. Saffiotti, U., and Shubik, P. The Role of Burning in Carcinogenesis. Brit. J. Cancer, 10: 54-56, 1956.

17. Sinnott, E. W., and Bloch, R. The Relative Position of CellWalls in Developing Plant Tissues. Am. J. Botany, 88: 607-17,1941.

18. Sparrow, A. H., Cuany, R. L., Miksche, J. P., and Schairer,L. A. Some Factors Affecting the Responses of Plants to Acuteand Chronic Radiation Exposures. Radiation Botany, 1:10-34, 1961.

19. Stonier, T. Labeling Crown Gall Bacteria with P32for Radio-autography. J. Bacteriol., 72: 259-68, 1956.

20. Szent-Gyorgy, A., Hegyeli, A., and McLaiighlin, J. A. CancerTherapy: A Possible New Approach. Science, 140: 1391-92,1963.

21. Wehnelt, B., Untersuchungen Ã¼berdas Wundhormon derPflanzen. Jahrb. Wiss. Bot., 66: 773-813, 1927.





FIGS. 1-4. Wound healing in KalanchoÃ«stems. All cross sections X 125. The wound edge is at the left of each figure. The path of thefleedle used in wound is indicated by the large arrow in Fig. 1. Epidermis is indicated by E, cortex by C, and pith by P.

FIG. 1. No wound stimulated divisions, scored 0.FIG. 2. Note the cell divisions parallel to the plane of the wound forming the wound cambium (W.C.), scored as 0.5.FIGS. 3, 4. Fully developed wound cambium, scored 1.0.

AUGUST 1966 1605



1966;26:1597-1605. Cancer Res Jacques Lipetz Healing and the Tumorigenic ResponseCrown Gall Tumorigenesis: II. Relations between Wound

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cancer research · cancer research volume26 august 1966 number8 [cancer research 26 part 1,...

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