D I S C U S S I O N S E C R E T A R Y ' S R E P O R T P . P . De n d y
Dept. of Radiotherapeutics, Cambridge
After thanking the Rapporteur for his excellent summary, Ca r l s o n
spoke briefly of the rationale behind their experiments and the interpretation which they put on the results. The earlier study of
Ga u l d e n and Pe r r y (1958) had shown that the mitotic progress of a grasshopper neuroblast could be stopped by treating one of the nucleoli with polychromatic u.v. It seemed advantageous to try to determine whether this resulted from a direct effect on the nucleolar proteins or, alternatively, an effect on the nucleolar R N A which might sub- sequently involve protein synthesis. The wavelengths 265 nm and 280 nm were therefore chosen for further investigation. A correction must be made when interpreting the results since the nucleolus probably contains about twenty times as much protein as R N A and a further correction which largely offsets the former allows for the fact that the relative absorptions of R N A and protein for u.v. light are in the ratio of approximately ten to one. At most of the mitotic stages studied, u.v. radiation at 265 nm is consistently more efficient in delaying mitosis than radiation at 280 nm and this seems to indicate that mitotic delay is mainly produced through an effect on the R N A though some direct effect on the nucleolar protein cannot be excluded.
Pi t t m a n asked Ca r l s o n if those cells with one large and one small nucleolus, in which the mitotic organizer still seemed to be functional since they survived the next division, had been followed through subsequent divisions. In effect, was it possible to detect any delayed radiation effect which could be correlated with the unequal division of nucleolar material ? Ca r l s o n said that many of the cells in their preparations had been followed for several days after irradiation and that the required information had probably been recorded. He was, however, unable to recall the answer to this question and did not have the necessary data with him. Vo n Dö h r e n stated that when their
325
experiments were extended beyond the first mitosis, the cells died.
He suggested that irradiation might block certain processes, thus making more energy available to those changes immediately prior to mitosis. In consequence, these changes would be speeded up.
Gl u b r e c h t pointed out that any action spectrum would be depend- ent upon the appropriate absorption spectrum at the site of irradiation.
The importance of graphs which show radiation efficiency plotted against equal absorbed doses of energy rather than equal incident fluxes at different wavelengths must be emphasized, and Gl u b r e c h t
asked both Ca r l s o n and Zi r k l e if they had any data on absorption characteristics. Ca r l s o n stated that in their experiments the fluxes quoted were for incident energy and they had no actual measurements of the amount of u.v. absorption which occurred before the beam reached the nucleolus. However, this absorption was kept to a mini- mum by confining the irradiations to those cells in which the nucleolus was within ζ μ of the quartz cover-slip. In Zi r k l e ' s experiments on spindle disappearance following u.v. microbeam irradiation of the cytoplasm the absorption spectrum of the cytoplasm had been found and it was unlikely that it would fit the action spectrum for spindle disappearance as well as a pure protein. Before leaving this topic
Gl u b r e c h t pointed out that in their laboratory low-energy micro- absorption spectroscopy can be used to measure absorption spectra for preselected microbeam fields in living cells.
Ja g g e r asked Zi r k l e if he had any information on the relation between effect on the spindle and the site of irradiation, and this was followed by a question from Ra j e w s k y who asked if there was any evidence for spindle restitution following the loss of birefringence.
In reply to the former question Zi r k l e stated that the site of irradiation was routinely selected to be as close as possible to the spindle fibres without actually irradiating any of the chromosomes, which are quite large in these cells, but in certain instances radiation directed well away from the mitotic apparatus and even at the outermost edge of the cell had resulted in a loss of birefringence. In answer to the second question, there has never been any evidence of restored birefringence after complete spindle destruction, even after several days. Cells in which the spindle had been only partly destroyed usually passed through anaphase, although this was not always a normal structure, and hence the spindle disappeared.
Fo r e r indicated that his experiments were designed to obtain
direct evidence regarding the role of the chromosomal spindle fibres
in anaphase chromosome movement. In this connection, five observa- tions must be emphasized:
ι. Area of reduced birefringence moves towards the pole whether or not the chromosomes move.
2. These areas move towards the pole at a constant velocity and both sides of the area move with approximately the same velocity.
3. If both a chromosome and an area of reduced birefringence move towards a pole along the same fibre or bundle of fibres their rates of movement are quite independent.
4. When u.v. irradiation stops chromosome movement, both members of the separating pair of chromosomes stop moving.
5. Chromosomes which are adjacent to an area of reduced bire- fringence are able to move normally.
Since extra-spindle irradiation affected neither the chromosome movement nor the birefringence, there must be a spatial association between the half-spindle and chromosome movement. However, the results summarized above indicate clearly that the biréfringent property of these fibres is not essential for chromosome movement and, if these fibres are responsible for the force which separates the chromo- somes, their optical anisotropy must be able to be affected quite independently of the force mechanism. Alternatively, we may search for another type of fibre which might be responsible for the force mechanism.
Sm i t h commented on the abnormally small percentage of giant cells and binucleate cells following nuclear sap irradiation. He pointed out that these experiments are still at a relatively early stage and, in consequence, the cell sample on which this percentage is based is small
—in absolute terms only five abnormal cells have been recorded in this category. It is therefore to be hoped that this anomaly will be resolved when more data are available. Pi t t m a n then asked Sm i t h if the cells which had become detached from the quartz cover-slip (a detachment which usually occurred when changing the nutrient medium) following u.v. microbeam irradiation of the nucleolus, had been collected and examined. He suggested that these cells might have reached a stage in mitosis in which they were easily teased from the quartz by the addition of new nutrient medium. In other words, is it possible that nucleolar irradiated cells exhibit enhanced mitotic activity in a synchronous manner and are thus detached from the quartz in greater numbers than the control cells ? In reply, Sm i t h stated that these detached cells had
not been analysed, but the above explanation was most improbable.
During the period between 12 and 24 h after irradiation many of the nucleolar irradiated cells showed visible signs of radiation sickness.
Since the most striking of these changes was a retraction of cyto- plasmic processes, presumably coupled with a reduction in the ability to adhere to the quartz, a loss of cells when the feeding mixture was changed was to be expected.
Zi r k l e congratulated Bu r n s and Pa t t e e on the design of their microbeam which can deliver a flux of X-rays more than 100 times greater than the flux which has been achieved previously for an X-ray microbeam. This apparatus is almost as convenient to use as a u.v.
microbeam.
In connection with the elaborate precautions adopted by Fo r e r
et al to allow for the change in focal length of the reflecting objective when a quartz cover-slip is interposed, Gl u b r e c h t indicated the advantage of an Ultrafluar transmission objective. This objective is achromatic from 240 nm to 700 nm when used in conjunction with a quartz cover-slip of specified thickness and the appropriate immersion medium.
Sm i t h then asked Be s s i s about the length of pulse and dosimetry for the laser microbeams and Re d h a r d t asked if the effects of a normal pulsed laser beam had been compared with the effect of a beam from a Q-switched laser. Be s s i s replied that for the Ruby and Neodymium lasers the impulses last about i o ~3 sec compared with a few nano- seconds for the Q-switched laser. Te r Po g o s s i a n added that a suitable method for absolute dosimetry is not yet available. Both Gl u b r e c h t
and Te r Po g o s s i a n thought it unlikely that the quanta of light from a laser (energy ~ 2 ev) would produce ions or radicals, but Te r Po g o s - s i a n commented that their production might be possible in theory for the Q-switch laser in which the very high-energy density will produce high potentials and these might cause some ionization.
Mo n t g o m e r y commented on the advantage of a flying spot micro- beam for producing very low radiation energies. This may be useful if the radiation dose is to be spread over a long period of time. The maximum energy is only o-18 χ i o ~7 erg/sec/cm2 and this can be reduced even further by a blanking spot. Ha y n e s pointed out that the annular type of aperture described by Mo n t g o m e r y enabled only a relatively small proportion of the membrane area to be irradiated. The possibility that the bubbling was not a membrane effect had not been precluded. Mo n t g o m e r y agreed with this but explained that it could
Ga u l d e n M . E . and Pe r r y R.P. (1958) Proc. nat. Acad. Sei., Wash. 44, 553.
not be a direct local effect at the site of irradiation. In reply to Ra p s o n ,
who pointed out that only an 'all or nothing' effect had been demons- trated and inquired about the effect of much lower doses, Mo n t -
g o m e r y outlined their future experiments. Using progressively lower microbeam doses, directed at various cell sites, it is hoped to find a site at which the radiation will lead to the formation of an abnormal mononucleate cell, a radiation effect which has already been observed following whole cell X-ray irradiation, thus identifying within the cell a site which acts as a mitotic trigger.
The discussion concluded with comments by Ur e t z and Gl u - b r e c h t on the difficulty of obtaining statistically reliable dose effect curves in microbeam experiments owing to the small number of cells that can be handled at any one time.
R E F E R E N C E
