NATURE. VOL. 220. OCTOBER 12. 1968

infected and uninfected cells is given in Table 3. I t is apparent that the presence of (DX DNA does not increase the amount of E. coli DNA that will stick to R F filters. These results also illustrate (1) the repro- ducibility of the annealing procedure, (2) the differ- ence in the amount of hybridizable DNA synthesized in extracts of infected and uninfected cells, and (3) that the amounts of input DNA (which are roughly the same in a,ll of the experiments reported here) are below the level rcquired to saturate t,he RF on the filter.

As a result of the discovery that the synthesis of pro- gcny (DX174 DNA proceeded by displacement of a plus strand from R F with the concomitant synthesis of a new plus strand2-5, two alterriative mechanisms wero cori- sidered. One envisaged de novo initiation of a new plus strand, resulting in intermediates which are shorter than the finished product. The other envisaged elongation of tho plus strand already present, thus giving rise to inter- mediates which are longer than the finished product. This lat,ter possibility was suggested (by W. Gilbert and D. Dressler) to make it possible for E. coli DNA polymer- ase (which is known to require a 3' OH to add on to) to catalyse the synthesis of single stranded QX DNA. Experiments are in progress both here a t Harvard and at the California Institute of Technology to determine the existence and relevance of long and short strands.

The experiments reported here wero intended to ask t,he question whether or not the E. coli DNA polymerase isolated by Kornberg was respor~sible for the synthesis of single stranded DNA in these extracts and hence presumably in vivo. The results indicate fairly clearly either that E. coli DNA polymerase does not catalyse the synthesis of hybridizable (DX DNA in the extract or that, if it does, it is not readily inhibitable by anti- bodies.

This result is compatible with the experiments rcportcd by Steinberg and me7 showing that a bacterium the ability of which to replicate DNA is temperature sensi- tive is also unable to support the contiriued synthesis of single stranded @X DNA if the (DX-infected cells are shifted from the permissive to the restrictive temperat,ure during the period of single stranded DNA synthesis. Yet extracts of this bacterium exhibit normal Kornberg enzyme activity at both temperatures7.

None of these results prove that the E. coli DNA polymerase isolated by Kornberg is not involved in the reaction. It is possible that the interaction of the enzyme with the (DX template alters its properties or that associa- tion with other components is required for certain types of DNA synthetic reactions, but not for all, and the presence or absence of these other coniporients affects the properties of the enzyme. In this connexion Hnh arid Helleiners have reported that oxpressiorl of DNA polymerase activity in extracts of QX-infected and unin- fected cells is different, though when they tried to purify the activity they were unable to find any indication of a now polymerase.

The results reported here prove that there is some difference between the reaction leading to the non-specific labelling of the bacterial DNA (which I believe reflects primarily attempts to repair the fragments of the bacterial DNA generated in the process of making the extract)' and the reaction leading to the synthesis of (DX DNA. The similarities of the characteristics of the in vitro synthesis of QX DNA reported in this and the preceding1 paper to tJhe characteristics of the in vivo process argue that the in vitro process in these extracts is a t least in part the same as the rlormal in vivo process. At present I favour the hypothesis that a bacterial enzyme plays a part in the synthesis of (DX single stranded DNA (both in eivo and in these extracts) but that this enzyme is not simply the enzyme isolated by Kornberg from E . coli.

I tharik Kathryn Brornstrlip for technical assistance and Dr Nicholas Cozzarelli for advice and samples of DNA polymerase and antiserum to DNA polymerase. This work was supported by grants from the US Public Health Service and the American Cancer Society.

Received August 5 ; revised August 26, 1068.

Denharat, D. T., and Burgess, A. B., Cold Sprzng Harbor Symp. Quavt. Biol., 33 (in the press).

Dresslcr, D. R., and Denhardt, D. T., Nature, 219, 346 (1968). T'indqvist, U. H., and Sinsheimer, R. L., J. Mol. Biol., 32, 2R5 (1068). Rnippcrs, R., Komano, T., and Sinsheimer, R. L., Proc. U S Nut . Acad. Sci.,

Rf l 577 (1RKX) - - , > - - - - , . Romano, T., Kni~pers, It., and Sinsheim~r, R. L., Proc. U S Nat. Acad. Sci.,

59,911 (1968). ' Denhardt, D. T., Biochem. Biophys. Res. Commun., 23, 641 (1966).

Steinberg, R. A,, and Denhnrdt, D. T., J. nlol. Biol. (in the press). Huh, T. Y., and Helleiner, C. W., Riochim. Biophys. Acta,155,107 (1968).

Chalones and Cancer In a number o f tissues it has been found that mi tot ic activity is controlled by specific mi tot ic inhibitors, called chalones. It is now shown that in four malignant tumours (epidermal carcinoma, granu- locytic leukaemia, melanotic and amelanotic melanomata) the chalones o f the tissues o f origin are stil l synthesized, although the chalone content is abnormally low, and that in all cases the tumours are stil l capable o f responding by mitot ic inhibit ion when the chalone content is artificially raised. It is also shown that when extracts o f normal skin o r o f melanomata (both o f which contain the melanocyte chalone) are injected for 5 days in to mice o r hamsters bearing large malignant melanomata, rapid tumour regression occurs and in some cases leads t o a complete cure.

Epidermal Chalone and Mitotic Control a glucocorticoid hormone4 inhibits epidermal mitosis in

in the V x 2 Epidermal Tumour a tissue-specific manner. There is evidence that it is this substance which, in terms of its intracellular coricciltm-

IT has been shown1 that normal mammalian epidermis tion, maintains the balance between cell production and produces and contains a substance, now called the epi- loss in the epidermis5-'j. In carcinogcnesis the basic evcnt dermal chalone2, which in the preserice of adrenalin3 and is clearly the disruption of this normal balance so that an

NATURE, VOL. 220. OCTOBER 12, 1968

unusually large mass of cells is able to accumulate6. The questiorl therefore arises whether in an epidermal tumour there is any change in the chalone mechanism. In particu- lar it is important to discover whether the cells fail to produce or retain normal amounts of the epidermal chalone or whether they fail to respond normally to any epidermal chalone that may be present. The results givcn here have already been briefly reporteds.

In devising the experiments it was necessary to use a transplantable tumour and to maintain it in genetically similar animals so that meaningful comparisons could bc made between the different experimental groups. The rabbit V x 2 epidermal tumour, originally derived from the Shope papilloma, was chosen; it is now a poorly differentiated, fast growing, squamous cell carcinoma which metastasizes early. Fragments were inoculated intramuscularly in the thighs of adult albino rabbits and all the experiments were performcd 2 weeks later. These tumours were capable of killing a rabbit in as little as 4 weeks.

In the first experiments, turnours were macerated and extracted in water, and after centrifugation the clcar supernatant was tested for the presence of the epidermal chalone. The test was made on pieces (about 3 mm2) of normal mouse ear epidermis maintained a t 37' C in a buffered saline medium with glucose and an oxygen gas phase7. In such an experiment a mitotic inhibition can only be ascribed to the epidcrmal chalone if it sat,isfies two criteria: it must be epidermis-specific and it must hc dependent on the presence of trace amonnts of adrenalin. When epidermis from freshly killed mice is placed in vitro it contains sufficient adrenalin to permit a powerful response to epidermal chalone in t,he culture medium, but within about 5 h this adrenalin is lost and the chalone loses its power to inhibit mitosiss. At this time a brief wash in a dilute adrenalin solution immediately restores the responsiveness of the epidermis to the chalorie.

The extracts of the V x 2 tumour satisfied these tests and it is therefore clear that the tumour cells contained a t least some epidermal chalone.

Tests were then made of the ability of the V x 2 cells to respond by mitotic inhibition to the epidermal chalone. Pieces (about 3 mms) of the outermost mitotic zone of the tumours were incubated for 4 h in saline medium in the same way as were the pieces of ear. The epidermal chalone, prepared from pig skin by N. V. Organon, Oss, The Netherlands, was in the form of a partially purified, 71-80 per cent ethanol precipitateg. Thc dosage used is expressed in mg of the original water soluble material obtained from the pig skin. About 40 yg of the 71-80 per cent ethanol precipitate is equivalent to 1 mg of the original material and is therefore described as 1 mg equivalent.

The results (Fig. 1) indicated that no mitotic inhibition was obtained with chalone (0.25 mg equiv./ml.; N, No. of tumours, = 35), adrenalin (2.5 yg/ml.; N = 34), or hydrocortisone (2.5 yg/ml.; N = 30) alone, or with chalone plus adrenalin (N=25) or chalonc plus hydrocortisone (N=36). Significant mitotic depressions of 11 per cent were obtained, however, with adrcnalirl plus hydrocorti- sone (N = 58) arid of 53 per cent with chalorie plus adrenalin plus hydrocortisone (N = 75). Thus tho importance of the stress hormones is again obvious, and it appcars that the small mitotic depression obtained with these hormones alone was probably due to their cooperation with the endogenous chalone of the tumour cells.

The action of the epidermal chaloric was also tested in vivo using 5,000 mg equiv. per rabbit injected sub- cutaneously, when the mitotic depression in the V x 2 tumours (N= 19) during the ncxt 4 h was about 70 pcr cent. In these circumstances, the chalone was evidently able to exert its antimitotic action in cooperation with the endogenous stress hormones.

The V x 2 cells clearly retain their ability to respond by mitotic inhibition to the epidernlal chalone, and so it

seems that their high mitotic activity may be related either to their failure to synthesize sufficient chalone or to their inability to retain what ehalone they do synthesize. I t is significant that in rabbits with large V x 2 tumours thc mitotic activity of the normal epidermis was found to be deeply depressed, evidently because of the large amounts of epidcrmal chalone that were escaping from the tumour into the body. This suggests that onc basic fault in the V x 2 cells may be that the cell wall is un- usually permeable to the chalone. An exactly similar conclusion has been reached by Ryt8maa and Kiviniemi (see following communication) in relation to the granulo- cytic chalone of chloroleukaemic cells.

Effect of epidermal chalone and stress hormones

Fig. 1. Mitotic act,ivity of pieces of V x 2 epidermal tumour maintained in vitro and treated with enidermal chalone. adrenalin and hvdro- c~~rt iaonr nloltr 2nd i l l vuri,;us c~oml~in:~tio~~s. 'With t l ~ r cor&bil~:;tiol,s c ~ i c~h:tlorre plllrj :rtlr~~rl:~lin . ~ n d chnlorrr ~ > l n n IrytIrr~corti8on~ nnt shoan

here, there was no mitotic deprpssion.

The question now obviously arises whether such chalones may be of practical value in cancer cherno- therapy to arrest or even to reverse tumour growth. Because of the relatively small supply of epidermal chalone and the relatively large size of a rabbit, however, it has not yet been possible to use the V x 2 tumour in such an investigation. Therefore, bccausc no trans- plantable epidermal turnours were available in such smaller animals as mice and hamsters, the problem has been approached using transplantable melanomata treated with the melanocyte chalone. The results obtained are described in the following communications.

We thank Mr B. Devlin, St Thomas's Hospital, London, for V x 2 tumours for the in vitro work and Mr M. Gough, University College Hospital, London, for facilities for the in vivo work.

W. S. BUI~LOUGH EDNA B. LAIJRENCE

Mitosis Research Laboratory. Birkbeck College, University of London.

Keceived July 1, 1968.

' Bullough, \V. S., and Laurence, E. R. , Proc. Roy. Soc., R, 151, .517 (1960). Biillough, W. S., B i d . Rev., 37, 307 (1962).

a Hullough, W. S., and Laurence, E. B., Proc. Roy. Soc., R, 154, ,540 (1961). Bullough, W. S., and Laurence, E. B., Cell Tissue Kiaut., 1, 5 (1968). BuIIough, W. S., Cancer Res., 25, 1683 (1965). Bullough, W. S., and Laurence, E. R., Advances in BioZoyy of Skin, VIT,

Carcinogenesis (edit. by Montagna, W., and Dohson, R. I,.) (?ergamon Press New York, 1965); Riillough, W. S., The E'zolfrtlor~ of D,~fferentza- tion (Academic Press, London, 1967).

' Bullough, W. S., and Laurence, E. R., Exp. Cell Res., 24, 289 (1961). Bullough, W. S., and Laurence, E. B., Exp. Cell Res., 38,176 (1964). Bullough W S. Hewett, (7 . L and Laurence, E. H., Exp. Cell Res., 36,192

(1964): Hbn6ius-Boldingh."~., and Laurence, E. B.,Europ. J. Riochem., 5, 191 (1968).