N4 virion DNA dependent-RNA polymerase: initiation sequences utilized by the enzyme on heterologous templates.

Bacteriophage N4 virion-encapsulated RNA polymerase, the enzyme responsible for transcription of the phage early RNAs, is unable to use duplex linear DNA as a template. In contrast to other RNA polymerases, the enzyme transcribes denatured N4 DNA with in vivo specificity. The promoter sequences for three sites of transcription initiation on the N4 genome have been determined and found to contain conserved sequences and two sets of inverted repeats. In order to define the minimal sequence requirements for N4 virion RNA polymerase activity, we have screened several heterologous DNAs, amounting to 64,328 bases, for their ability to support transcription. Several sequences allowing specific initiation were found. Their location, properties and the relation to N4 virion RNA polymerase promoters are discussed.

The effect of neonatal treatment of male mice with antiandrogens and of females with androgens on the development of the os penis and os clitoridis.

The os penis in mice and rats is composed of a proximal intramembranous and endochondral osseous element and a distal cartilaginous, ossifying element. Female mice, but not rats, have a small os clitoridis which corresponds to the intramembranous part of the proximal element of the os penis. In mice of either sex a dense mesenchymatous formation ventral to the urethra is the anlage for the bones of the external genitalia. In the early postnatal period the proximal part of the os penis develops as bone at the outer and as cartilage at the basal end of the anlage, while in females a minute focus of ossification differentiates into the small os clitoridis without passing through a cartilaginous phase. The distal element of the os penis is formed later than the proximal rod and grows at a slower rate. Neonatal treatment with an antiandrogen inhibits the increase in size and calcification of the os penis. Neonatal castration is an even more effective inhibitor. Neonatal treatment with testosterone or dihydrotestosterone, but not with oestradiol, stimulates the growth of the bony proximal os clitoridis, but induces only a rudimentary collagenous distal element. The differences between mice and rats in the response of the tissues of the clitoris to androgenic treatment are discussed, particularly as regards the differentiation of proximal and distal elements.

The effect of growth hormone, insulin and alloxan-induced diabetes on carcinogenesis in the genital tract of intact and castrate female rats.

Castrate female rats given weekly applications of DMBA to the genital tract and treated additionally with growth hormone, insulin or alloxan (to induce diabetes) are heavier and have more sarcomatous and epithelial cervico-vaginal neoplasms than intact animals under the same experimental conditions. Promotion of carcinogenesis and gain in body weight are independent phenomena caused by castration in the medicated rats. Growth hormone is most effective in enhancing body weight in all animals, but least as regards tumour formation. It reduces the incidence of sarcomas in intacts, but raises that of epithelial neoplasms, and promotes both types of neoplasms in castrates. The highest incidence of cervico-vaginal epithelial and sarcomatous tumours occurs in spayed diabetics.Squamous celled epitheliomas of the vulva are not affected by castration or additional medication, while basal celled neoplasms tend to be more frequent in intacts than in castrates and particularly numerous in intact failed diabetics. Vulval sarcomas are usually rare but are increased in numbers in diabetic and in insulin treated intacts.Granular myoblastomas of the cervico-vaginal tract occur in intacts only and particularly in diabetics and those medicated with growth hormone or insulin.

The effect of variation in carcinogenic dosage on the induction of tumours in the dorsal and vulval skin of female rats.

The response to 5, 10, 20 or 40 weekly paintings with DMBA of the dorsal and vulval skin in intact and castrate rats is compared. Squamous and basal celled tumours appear faster in the dorsal than the vulval region with 5, 10, or 20 paintings, but at the same rate with 40 doses. The rate of induction of epithelial tumours is optimal with 20 applications dorsally, but increases with dose at the vulva. Progression of malignancy of squamous celled tumours is greater and faster in the dorsal than in the vulval region. For basal celled neoplasms of the vulva there is a peak value in malignant conversion at 20 doses, but otherwise there is no consistent difference in the pattern at the two sites. Castration reduces the incidence of basal celled tumours of the vulva in rats painted weekly for life, but does not affect the incidence of epithelial tumours of the skin. Sarcomas occur in 29% of rats in the dorsal region, but in only 0·4% at the vulva. Sarcomatous changes in the stroma of epitheliomas are also more frequent in the dorsal skin. Local factors rather than variation in individual sensitivity account for the differences with region in the carcinogenic response as shown by their persistence in rats treated simultaneously at both sites.

The influence of carcinogenic dosage and of sex on the induction of epitheliomas and sarcomas in the dorsal skin of rats.

The effect of varying the numbers (4, 5, 10, 20 and 40) of weekly applications of DMBA to the dorsal skin of intact and castrate male and female rats on the induction of basal and squamous celled epitheliomas and of sarcomas has been investigated.Basal celled tumours originate mainly in hair follicles and squamous celled neoplasms in the interfollicular regions of the epidermis and differ in their progression to malignancy. Penetration of the panniculus carnosus is neither a sufficient nor necessary criterion of malignancy since growing hair follicles pass through the muscle layer and carcinomas and sarcomas which are still confined to the dermis, spread along the perineural lymphatics and metastasise to the lungs.Sex and castration do not affect carcinogenesis of epitheliomas in the dorsal skin at any dose level. Significantly more sarcomas result from 20 weekly paintings in male than in female or castrate rats.The induction period for all tumour types is shortened in sensitive individuals only by an increase from 5 to 10 weekly applications. For less sensitive animals the rate of oncogenesis is accelerated with number of administrations up to 20, but slowed down from this level by 40 paintings. The optimal dose for speed of induction of all tumour types, for maximal yield of basal celled epitheliomas and for that of sarcomas in male rats is 20 weekly applications.THE PROGRESSION TO MALIGNANCY VARIES WITH TUMOUR TYPE: it is fast for sarcomas and slow for basal celled neoplasms. Of the 336 rats at risk only 1% have fibromas or other precursor lesions, while 40% have sarcomas; animals with squamous celled papillomas account for 12%, but those with carcinomas for 66%; there are, however, 64% of rats with basal celled papillomas and only 9% with carcinomas.The optimal dose phenomenon in carcinogenesis is discussed.

Sex difference and carcinogenic dosage in the induction of neoplasms in salivary glands of rats.

At low concentrations of DMBA (½% and 1%) twice as many sarcomas and carcinomas of the salivary glands are induced in male as in female rats. Additional oestrogens reduce neoplasms in males by one half while testosterone doubles them in females. The sex difference disappears at the higher dose levels of the carcinogen (2%).Females are more sensitive than males to the toxic effects of DMBA, though less sensitive to the carcinogenic action.Carcinomas rise to a single peak within 240 days while sarcomas appear as late as 770 days with secondary and tertiary peaks. This difference in pattern of induction may be due to the formation of a fibrous capsule separating persisting DMBA-deposits from the epithelial structures and thus protecting them from carcinogenic risk.

The effect of thyroactive substances on the induction of cervico-vaginal and vulval tumours in castrate rats at various levels of carcinogenic treatment.

Medication with L-thyroxine or methylthiouracil of castrate rats painted weekly 5, 10, 20 or 40 times with DMBA does not alter the order of thresholds for carcinogenesis which increases from that for cervico-vaginal epitheliomas via squamous celled and basal celled vulval tumours to cervicovaginal sarcomas. Methylthiouracil lowers the threshold for basal celled vulval neoplasms.Sarcomas reach a peak of 25% with 20 doses of DMBA in non-medicated rats, but rise to 90% and at a faster rate in animals given either of the thyroactive drugs with further carcinogenic treatment.The optimal dose phenomenon for cervico-vaginal epitheliomas, i.e. a significant fall with continued painting from a peak reached by 5 to 20 doses of DMBA, is not affected by medication with methylthiouracil or L-thyroxine.Thyroactive compounds accelerate the formation of squamous celled vulval tumours which reach a maximum with 20 DMBA paintings; the total incidence as well as the proportion of carcinomas to papillomas falls with further treatment.Methylthiouracil promotes formation of basal celled vulval tumours at low dose levels, but inhibits it at the highest. In medicated as in non-medicated rats the induction of basal celled tumours of the vulva follows an optimum dose pattern.The optimal dose phenomenon and the effect of thyroactive compounds on the tissue-specific sensitivity to carcinogens are discussed.

The influence of thyroactive substances on the induction of cervico-vaginal tumours in intact and castrate rats.

The effect of the administration of L-thyroxine and of methylthiouracil alone, together, in combination with stilboestrol or in the perinatal period on the induction of cervico-vaginal tumours by weekly local applications of DMBA was investigated in intact and castrate rats and compared with carcinogenesis in animals not additionally treated.In intact rats the rate of sarcoma induction is accelerated by methylthiouracil, delayed and reduced by methylthiouracil plus L-thyroxine and delayed by perinatal injection of either L-thyroxine or methylthiouracil. In castrates sarcoma induction is accelerated and increased by L-thyroxine, methylthiouracil and by combination of either substance with stilboestrol; it is accelerated but not significantly increased by combined treatment with the thyroactive compounds.The incidence of epithelial neoplasms is accelerated and increased in intacts and in castrates by methylthiouracil. This effect is slightly reduced in intacts but potentiated in castrates by additional stilboestrol treatment as well as by administration of L-thyroxine plus methylthiouracil.The incidence of sarcomas is significantly greater in intact than in spayed rats not additionally treated, greater in castrates than in intacts given L-thyroxine ± stilboestrol and not significantly different in intacts and castrates with any of the other additional medications. For epithelial tumours the incidence is low and similar in both groups without additional treatment, greater in spayed than intact animals given methylthiouracil plus stilboestrol or plus L-thyroxine.The influence of the thyroactive compounds on the induction of epithelial and sarcomatous tumours is not correlated with their effect on gain in body weight nor on growth of the stroma and of the epithelium of the vagina, cervix and uterus. Changes induced in the thyroid gland and the hypophysis are not correlated with those on carcinogenesis.Central and local factors may account for the differential response in carcinogenesis of intacts and castrates as well as of the epithelial and connective tissue of the cervico-vaginal tract to medication with thyroactive compounds.

The effects of increased numbers of carcinogenic treatments on the induction of cervico-vaginal and vulval tumours in intact and castrate rats.

The effect of 5, 10, 20 or 40 weekly local applications of DMBA on the induction of cervico-vaginal epithelial and sarcomatous tumours and on that of squamous celled vulval neoplasms was investigated in intact and castrate rats. The threshold dose increases in the following order: epithelial cervico-vaginal tumours of castrates, followed by those in intacts and by squamous celled vulval tumours and lastly by sarcomas in castrates and intacts.The incidence of sarcomas levels off at about 25% after 20 doses in spayed rats, but increases to 70% with dose in intacts. All sarcomas appear between 200 and 400 days. The incidence of vulval neoplasms increases and the duration of the induction period decreases with dose.Significantly more cervico-vaginal epithelial tumours occur with 5 to 20 paintings than with further application of DMBA. Their peak value is 60% in castrates and 20% in intacts. Castration promotes the progression of vulval papillomas to carcinomas. The sensitivity to carcinogenic stimulation is thus tissue specific and also subject to modification by hormones.While epithelial tumours are multifocal and pass through well-defined intermediate stages (radication, papillomas, microcarcinomas) to full malignancy, the early stages of sarcoma formation are rarely detected and ill-defined. For epitheliomas and sarcomas "invasion" is a criterion of malignancy only if invading cells have acquired "xenoplasia", i.e. the ability to grow in new environments. This capacity increases progressively and its initial lack accounts for the discrepancy between the incidence of embolism and that of metastatic deposits.

The effect of oestrogens, testosterone and progesterone on the induction of cervico-vaginal tumours in intact and castrate rats.

PIP: This study investigates the carcinogenic effects of castration, stilbestrol, progesterone, testosterone, and combined estrogen and progesterone treatment on Lister strain rats. While castration seems to reduce the number of sarcomas in the cervico-vaginal tract when induced by 9,10-dimethyl-1,2-benzanthracene (DMBA), estrogens in doses sufficient to enlarge to normal size the castrate uterus inhibit the formation of sarcomas in intact rats, and fail to promote that in castrate animals. On the other hand, stilbestrol in doses insufficient to restore to normal the atrophy of the uterus, promotes and accelerates the formation of sarcomas and of epithelial tumors of the cervico-vaginal tract in castrate but not in intact rats. Progesterone in intact rats retards the induction of sarcomas but promotes papillomas of the cervix and vagina. Combined with estrogens, progesterone restores the rate of sarcoma formation to the level of intact rats. In both intact and in castrated rats testosterone seems to lengthen the induction period of sarcomas or to increase the formation of epithelial tumors; these effects are reduced when testosterone is combined with stilbestrol. Cholesterol increases the formation of sarcomas and epithelial tumors in castrate rats. Epithelial tumors as well as sarcomas arise at about the same time and rate in castrate animals given intermittently stilbestrol per os. The rate of tumor formation is greater than that in intact animals without additional treatments. The carcinogenic effects of estrogens, progesterone, cholesterol and testosterone are not correlated with the effect of the same substances on the normal tissues of the uterus, vaginal stroma, or other target organs.^ieng