J. J. Trentin

The Quest for Human Cancer Viruses. A New Approach to an Old Problem reveals Cancer Induction in Hamsters by Human Adenovirus.

A new approach to the important but difficult task of revealing possible human tumor viruses has been presented in this article. By systematic testing of already known human viruses for oncogenic properties, it was found that in hamsters injected intrapulmonarily with tissue culture fluid of human type 12 adenovirus within 24 hours after birth there was a very high incidence of malignant tumors at the site of injection in from 1 to 3 months. The tumorinducing activity was not lost by filtration through Selas 02 filters or by tissue culture passages in HeLa cells. Tumors thus induced grew in, and killed, a high percentage of the unconditioned young adult hamsters into which they were transplanted. No such tumors occurred in hamsters injected with control tissue culture fluid or with culture fluids of the other viruses tested, or in control breeder hamsters. The possibility that contamination with polyoma virus and simian virus 40 might be responsible for the tumors induced was specifically excluded by a variety of tests. The possible involvement of still other, as yet unknown, contaminant viruses was excluded by a positive association of the tumor-inducing ability with the adenovirus content. Of eight human sera tested, only those four which neutralized the adenovirus-type cytopathic effect also neutralized the tumor-inducing effect. Of 700 human sera tested, 26 percent contained CPE-neutralizing antibodies for type 12 adenovirus at titers of 1:4 and higher (23).

Mortality and Skin Transplantability in X-Irradiated Mice Receiving Isologous, Homologous or Heterologous Bone Marrow.∗†

Summary The degree and duration of protection afforded by isologous, homologous and heterologous sources of bone marrow administered after lethal doses of x-irradiation in mice was directly proportional to the closeness of genetic relationship of the marrow donor to the irradiated mice. Uninijected controls were all dead by the 12th day. Mouse marrow from the same strain or from an F1 hybrid of the irradiated strain, or from a foreign strain, all gave excellent and essentially equal protection as late as 21 days post-irradiation. While mice receiving marrow from the same strain remained well protected beyond 100 days, those receiving marrow from a foreign strain showed considerable late mortality between the 21st and 60th days. Mortality in mice receiving F1 hybrid marrow was intermediate at 60 days. Guinea pig and rabbit marrow elicited a very low degree and duration of protection. Rat marrow permitted 55% of the irradiated mice to survive beyond the longest survival of the untreated controls. The duration of protection however was short, only 6% at 60 days. Irradiated mice protected with marrow from an F1 hybrid of the irradiated strain showed a high degree and long duration of tolerance of skin homografts from the F1 hybrid or from the other parent strain. Irradiated mice protected with foreign strain mouse marrow showed a high degree and long duration of tolerance for skin homografts from the same foreign strain. Irradiated mice protected with marrow from the same strain did not tolerate skin homo-grafts from a foreign strain or from an F1 hybrid.

TOLERANCE AND HOMOLOGOUS DISEASE IN IRRADIATED MICE PROTECTED WITH HOMOLOGOUS BONE MARROW

The protection of mammals of several species against otherwise lethal doses of whole-body X irradiation by postirradiation transfusion of bone marrow is a well-established phenomenon. Protection is associated with reseeding and repopulation of the hematopoietic system with cells of marrow-donor origin, regardless of whether protection is achieved with isologous (same genotype), homologous (different genotype, same species), or heterologous (different species) bone marrow. Protection with homologous or heterologous bone marrow confers tolerance of skin grafts of marrow-donor antigenic type (that is, from the marrow donor or its parent strains if the marrow donor is an F1 hybrid), as well as of the transplanted Mice protected with isologousmarrow arenot rendered tolerant of homologous skin? 3 , Mice protected with homologous marrow may or may not show tolerance of skin from a third homologous train.^ Mice protected with rat marrow may accept rat skin while simultaneously rejecting homologous mouse skin. TABLE 1 presents the results of an experiment designed to determine the lowest dose of whole-body X irradiation, followed by marrow from an F1 hybrid, that would result in tolerance of skin homografts from the foreign parent strain.6 Only a t the high dose levels of LDYo and above was tolerance observed. At the high sublethal dose of 550 r, skin grafting could not be performed, since most of the mice died within 2 or 3 weeks after irradiation. The same dose of homologous F1 marrow essential for survival after a lethal dose of irradiation resulted in death when given after a high sublethal dose of irradiation! Death was preceded by a precipitous loss of body weight and reduced circulating red and white blood cell levels as compared to mice receiving the same marrow after 770 r. Evidence will be presented later indicating that this high sublethal zone mortality is the result of recovery of the ability of the host to reject the temporarily grafted homologous marrow. Main and Prehn5 found that 96 per cent of irradiated mice accepted skin homografts from the marrow donor after 600 r and 29 per cent after only 300 r. It is probable that the difference in results is related to the difference in genetic material used. The skin donor and recipient strains of TABLE 1 are of different histocompatibility-2 (H-2) genotype. Main and Prehn used two strains of the same H-2 genotype. The H-2 locus is the strongest of the loci determining histocompatibility in the mouse? What is the nature or mechanism of the tolerance of homologous skin induced by irradiation protection with bone marrow of skin donor type? To use the terms of Billingham et uZ.,B is it the result of a central failure, or of a peripheral failure (afferent or efferent), of the immunological response that constitutes the

Cancer Induction in Hamsters by Human Type 12 Adenovirus. Effect of Age and of Virus Dose.

Summary The susceptibility of newborn hamsters to induction of sarcomas at the site of injection of human type 12 adenovirus decreases rapidly with increasing age at time of injection. For a given age at the time of injection, the incidence of tumors induced is directly proportional to the dose of virus injected. The 50% tumor-inducing dose in hamsters injected when less than one day of age lies between 0.5 and 50 MTCID100 of adenovirus under the conditions of these experiments. Increasing the dose of virus to 1000 MTCID100 produced one tumor among 8 hamsters injected as late as 14 days of age, but none in 3-week or older hamsters.

Induced tolerance and homologous disease in x-irradiated mice protected with homologous bone marrow.

Summary CBA mice protected against an otherwise lethal dose of irradiation with isologous CBA bone marrow showed normal rejection of skin grafts from the Cb strain. Irradiated CBA mice protected with Cb or (Cb x CBA) F1 hybrid marrow were tolerant of skin grafts from the Cb strain. Irradiated CBA mice protected with a mixture of marrow from the Cb and CBA strain were not tolerant of skin grafts from the Cb strain. Late mortality (21 to 60 days post-irradiation), after good early protection (21 days) against doses of whole body X-irradiation above the LD100, did not occur in mice protected with isologous bone marrow (where neither the host nor the donor can react immunologically against each other). It occurred in mice protected with homologous marrow from a foreign strain (where both the host strain and the donor strain can potentially react immunologically against each other). It did not occur in mice protected with marrow from an F, hybrid of the irradiated strain (where the host strain can potentially react against the donor strain but the donor strain has no potential to react against the host strain). It occurred in some but not all combinations of irradiated F1 hybrids protected with marrow from a parent strain (where the host has no potential to react against the donor strain but the donor strain can potentially react against the recipient strain). The terms “homologous disease”and “heterologous disease”are proposed to designate the complications specifically attributable to successful transplantation of homologous or heterologous, as opposed to isologous or autologous, bone marrow or lymphoid tissue into a tolerant host.

Effect of X-Ray Dose on Mortality and Skin Transplantability in Mice Receiving F1 Hybrid Marrow.∗:

Summary The 21-day LD50 under the conditions of X-irradiation used is approximately 600 r. Irradiation of CBA mice followed by intravenous administration of bone marrow suspension from Cb x CBA F1 hybrids resulted in little or no 21-day mortality at 110, 330, 660 and 770 r, but in almost 100% mortality at 550 r. Homografts of Cb skin into the surviving mice were accepted by the mice at 660 and 770 r dose levels but not by mice at 0, 110 and 330 r dose levels. None of the marrow-treated mice at 550 r survived long enough to be skin grafted.

Cancer Induction in Hamsters by Human Type 12 Adenovirus. Effect of Route of Injection.

Summary Tumors were induced at the site of injection of human type 12 adenovirus into newborn hamsters by each of the intrapulmonary, intrapleural, intraperitoneal, intravenous and subcutaneous routes, leading to death within 29 to 108 days. Whereas at higher doses of virus the subcutaneous route of injection was as effective as the other routes, at lower doses of virus it appeared less effective. For the intracranial route of injection, no local tumors were observed but one such hamster developed multiple abdominal tumors, and 3 developed hydrocephalus. Of 35 hamsters with tumors at the site of injection by various routes, remote tumors in the liver also developed in 10. Similar liver tumors were also found in 4 intravenously injected hamsters without a tumor at the site of intravenous injection. Of 7 hamsters administered virus by intranasal instillation, none have died of tumors in approximately 9 months to date.

Characteristics of Human Adenovirus Type 12 Induced Hamster Tumor Cells in Tissue Culture.

Summary Characteristics of a human adenovirus type 12 induced hamster tumor serially propagated in vitro are described. These include small cell size, epithelioid appearance, rapid growth rate, resistance to superinfection with A-12, and transplantability to weanling hamsters. These cells grew either as monolayers or as balls of aggregated cells detached from the glass, depending on whether calf serum or horse serum was added to the Eagles medium. Attempts to demonstrate virus activity by subculture of supernatant fluids and lysed cells into HeLa cells, mixed culture with human and hamster cells, electron microscopy, and inoculation of newborn hamsters with irradiated tumor cells were negative.

Persistent Infection of Human Cells (HeLa) with Adenovirus Type 12.

Summary The establishment of an adeno-virus type 12 cell carrier system is described. Characteristics of the carrier cultures include an initial dependence on inhibitor or antibody in human serum, an enhanced rate of growth after the fifth day as compared to uninfected cells, a relatively high percentage of infected cells, but a low percentage of detectable virus yielding cells, and relative resistance to super-infection. Secondary mechanisms responsible for the carrier state may include the selection of resistant cells, interference by non-infectious virus and inter-feron.