INTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, VOL. IX, 473-478 (1975)

Ultraviolet Carcinogenesis

PHILIP ROSEN* Biology Diuision, Oak Ridge National Laboratory,? Oak Ridge, Tennessee 37830, USA, and Department of Physics and Astronomy, University of Massachusetts, Amherst,

Massachusetts 01002, USA

Abstracts

A theory of uv carcinogenesis is described in which damage to DNA in the form of pyrimidine dimers occurs in a critical operator region of the genome that controls the synthesis of divisional proteins. There is a chance that a mutation will occur in the region of the operator either during replication or during postreplication repair, so that unchecked growth will be passed on to daughter cells. Comparison with the experiments of Hart and Setlow (1973) indicates that agreement is semiquantitative.

Une thCorie de la carcinog6ncse uv est dCcrite, dans laquelle I’ADN dans la forme de dimbres de pyrimidine est atteint dans une rCgion d’opCrateur critique du genome qui contrble la synthkse des protkines divisionnelles. I1 y a une chance qu’une mutation surgisse dans cette r e g i o n 4 soit pendant la rCplication soit pendant la postrCplication, ce qui pourrait entrainer une croissance non contr6lte des cellules filiales. Une comparaison avec les exp6riences de Hart-Setlow (1973) indique un accord semi-quantitatif.

Eine Theorie fur uv-Carcinogenes wird beschrieben, in welcher DNA in der Form von Pyrimidindimeren beschadigt wird in einem kritischen Operatorbereich des Genoms, das die Synthese von divisionellen Proteinen kontrolliert. Es gibt eine Moglichkeit dass eine Mutation in diesem Operatorbereich entweder wahrend der Replikation oder der Postreplikation entsteht, so dass unkontrolliertes Wachsen zu den Tochterzellen iibertragen wird. Ein Vergleich mit den Hart-Setlow (1973)-Experimenten deutet eine semi-quantitative Ubereinstimmung an.

1. Introduction

Recent biochemical evidence (Setlow, Regan, German and Carrier [ 11; Cleaver [2]) indicates that damage to DNA is involved in carcinogenesis. DNA excision-repair activity was found to be deficient in skin cells from xeroderma pigmentosum patients. uv-induced thymine-containing dimers in these patients are not excised. Recent reviews of photoproducts in uv-irradiated DNA are given by Varghese [3] and Rahn [4].

Recently evidence has been obtained (Hart and Setlow [5]) that pyrimidine

* This investigation was supported in part by an NIH Fellowship (1F03 CA 52956-01) from the

t Operated by the Union Carbide Corporation for the U.S. Atomic Energy Commission. National Cancer Institute.

473 @ 1975 by John Wiley & Sons, Inc.

474 ROSEN

dimers are responsible for tumor induction in the fish Poecelia forrnosa. Fish contain photoreactivating enzyme, an enzyme that monomerizes dimers but does not affect other products in DNA, and hence are a good organism for investigation of the molecular basis of uv-induced neoplastic transformation. Homogenates of tissue from various organs were exposed to 254-nm radiation. Portions of these homogenates were injected into fish from the same clone and the percenlage of fish developing tumors was measured. If, however, a homogenate was illuminated with photoreactivating light (320-450 nm) before injection, the yield was reduced by an amount dependent on the illumination time.

In this paper a quantitative theory of uv carcinogenesis is presented. Previously I suggested that initiation of cancer may be due to DNA damage in an operator region of a functional operon which is interconnected with mitotic operons [6]. I would like to expand this idea quantitatively for uv car- cinogenesis.

Most uv-induced effects in DNA involve thymine, even though uv light is absorbed by all four bases (Eisinger and Shulman [7]). Instead of trying to calculate the average number of defects per unit length of DNA as a function of uv dose from first principles (at present an impossible task), we will use experimentally determined values. First let us look at the relative efficiency of formation of various photoproducts in E. coli. At 254-nm wavelength (Rahn [4]; Rahn and Landry [8]; and Rahn, Landry and Carrier, [9]), if the relative efficiency of production of the dimer TT is unity, then the dimer CT is 0.2, the dimer CC is 0.2, single-strand breaks are 0.01-0.001, double-strand breaks are <0.001, and cross links are 0.01-0.001. Because of these results we will put our emphasis on the pyrimidine dimers.

2. Average Number of Dimers per Unit Length

In the review article by Setlow and Setlow [lo], the number of dimers formed per genome in both E. coli (2.5X lo9 daltons) and mammalian ( 3 X 10l2 daltons) cells is given. For both these cases the number of dimers per base pair per erg/mm2 at 254nm is about (see also Kondo, Ichikawa, Isher and Kato

If indeed the distribution of dimers is random (Rahn [12]) and if their formation is a rare event following Poisson statistics, we should have as the probability of n hits in a region of length L, P,:

[111).

where G(D) is the average number of dimers per unit length. The probability of at least one hit in a region of length L is

ULTRAVIOLET CARCINOGENESIS 475

At low dose with ii(D)=KD where K is a constant we have

(3) PZ1 = LKD with K= Base Pair mmz dimers I=

3. The Size of the Critical Region

Consider an operator region which controls the synthesis of divisional proteins. Such controls, as well as parallel controls for replication, were recently discussed by Jones and Donachie [13]. If there is any dimer in the operator, the repressor associated with this operator may no longer bind. It is also possible that there is a dimer in the gene coding for the repressor (the i gene). A mutation in the repressor protein might prevent binding to the operator. For the moment we will consider this effect as secondary since the repressor has general binding to DNA but the operator sites are highly specific (Gilbert [14]). We cannot rule out a mutation in the i gene because its size is about 1000 base pairs (B.P.). The length of the i gene multiplied by an efficiency factor for derepres- sion might be an effective target length.

Then, assuming that DNA replication controls are normal, we should have continuing synthesis of divisional proteins and, if the damaged region does not lead to a mutation in the daughter cells in the same critical region, we would not have uncontrolled growth. Thus we not only require damage but subsequent mutation in the critical region.

The size of the lac operator is given by Reznikoff [lS] as 2 1 2 B.P. and less than 75 B.P. long. Gilbert [14] gives a value of 10-20 B.P. However, Watson [16] gives a value of approximately 20 B.P. for the lac operator region. We will assume here a critical length of 20B.P.

4. Mutations in Replication and Postreplication Repair

When cells of excision-defective strains of E. coli are exposed to uv- radiation, a mutation caused by a pyrimidine dimer can arise-either as an error introduced into the daughter strand as an unexcised dimer passes through the replication point or as an error introduced into the DNA in the course of postreplication repair. Howard-Flanders [ 171 has summarized the evidence that ultraviolet survival in E. coli is promoted by a recombinational mechanism which operates independently of excision repair.

If an error does not occur in replication itself, then an error can arise as follows: DNA synthesis takes place following the damaged template DNA until a dimer is reached. Synthesis then skips over the dimer with the resulting formation of a gap in the newly synthesized DNA (discussed in Setlow and Setlow [lo]). The gaps are opposite to the dimers. Upon incubation of these cells, the gaps become filled in and an error is possible. The average gap size is

476 ROSEN

3 X 10’ daltons or 900 bases. In nonexcising human cells there are gaps of 2 . 4 ~ 10’ daltons (lo3 nucleotides) which are filled in by postreplication (Buhl, Setlow and Regan [18]).

Either in replication or in the process of gap-filling, a mutation may occur. Witkin [19] gives the frequency of uv-induced mutations in E . coli. The frequency of mutation, f, per unexcised dimer is

(4) 1 f = ~

2x 103

5. Tumor Initiation

We are now ready to combine the results in the model above to calculate the number of cells capable of initiating tumor formation. Given N cells which are subject to a dose (D) of uv-radiation and which undergo either postreplication repair or replication past a dimer (before it is excised) in the animal, then, assuming there is no immunological rejection of a damaged cell, the average number of initial tumor cells, filTC, is

- ( 5 ) NITc=Nefi . Pzi * f

where N e ~ is the number of surviving cells out of N, or

(6)

low dose we have

- NIT,= Near 1 -exp (-Lii(D))lf

We are assuming that every initial tumor cell formed is allowed to grow. At

(7) NIK = Ne&KfD

The last equation is linear in the region where N,tf is hardly dose-dependent. Such factors as f and operator size should be dependent on the type of species used as well as on the type of tissue.

In a group of Nef live cells containing NIT, cells on the average, we have Poisson statistics. Thus the probability of a group of cells containing n initial tumor cells, W,,, is

(8)

The probability

(9)

e-AqN,TC)” W,= n!

of at least one initial tumor cell is

w - l - e - N I T C 2 1 -

- For small fil,,, Wz 1 = NIT,.

The data of the experiment by Hart and Setlow [ 5 ] give the result for liver

homogenates of 5 x 10’ cells and an incident dose of lo3 erg as 15% of injected mm2

ULTRAVIOLET CARCINOGENESIS 477

fish developing tumors. The incident dose of 10 33 is an average fluence of

- lo2 (Hart and Setlow, private communication). For D = 10' 3 and

L = 20 B.P., Equation (7) yields

mm2 er

mm

NeE N The fraction of viable cells - will vary greatly as one goes from species to

species. This is so because lethal target lengths in DNA are variable (Kondo et al. [ll]). We shall therefore leave the result (7) for RrTC in the form

(10) RITC = rNLKfD

where r is the fraction of N cells remaining viable. For r=0.5 in the above numerical example, NIT,= 0.25 and the probability of finding at least one initial tumor cell is about 0.22. For r = 1, RITc= 0.5 and W Z 1 =0.38 (the observed value was 0.15).

To be complete we must correct Equations (7) and (10) for the possibility of excision repair. This can be done by multiplication by the factor (1 - R) on the right-hand side. R is the reparable fraction of dimers. Thus, if all dimers are reparable, there are no tumors. We have been assuming that R is very small for the fish in the Hart-Setlow experiment.

6. Conclusion

I have outlined a theory of uv carcinogenesis in which damage to a critical region of DNA occurs (most likely in the form of pyrimidine dimers), which in turn leads to a mutation in the region during either replication or postreplication repair. It is essential that'the damage remain unrepaired at least initially, and that tumor cells not be destroyed by some immunological mechanism. I have conjectured that the critical region of the genome be an operator region controlling divisional proteins.

Acknowledgment

The author acknowledges the aid of R. B. Setlow, P. Swenson, and R. W. Hart of the Biology Division of Oak Ridge National Laboratory, without whom this work might not have come to fruition.

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131 A. J. Varghese, in Photophysiology, Vol. 7, A. C . Giese, ed. (Academic Press, New York, 1972), p. 207.

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[15] W. S. Reznikoff, Ann. Rev. Genet. 6, 133 (1972). [16] J. D. Watson, in Molecular Biology ofthe Gene (W. A. Benjamin, New York, 1970), p. 448. [17] P. Howard-Flanders, Ann. Rev. Biochem. 37, 175 (1968). [18] S. N. Buhl, R. B. Setlow and J. D. Regan, Int. J. Radiat. Biol. 22, 417 (1972). 1191 E. M. Witkin, Ann. Rev. Genet. 3, 525 (1969).

University Press, New York, 1970), p. 946.

Received October 14, 1974.