Basic studies of cryochemotherapy in a murine tumor system

The combined effect of cryosurgery and anticancer drugs (cryochemotherapy) was studied in an experimental B16 melanoma/BDF1 tumor system. Vascular volume and vascular permeability after cryosurgery of normal skin and the tumor were measured by using 51Cr-labeled red blood cells and 125I-labeled serum albumin. The vascular volume and vascular permeability of both the normal vessels and the tumor vessels greatly increased immediately after cryosurgery, and their vascular volume decreased to less than the normal level within a few hours. However, the tumor vessels showed less dilatation and increase in permeability than the vessels of normal tissue. There was a difference in functional characteristics in response to cryoinjury between the normal vessels and the tumor vessels. The anticancer drugs, peplomycin and adriamycin, were administered intraperitoneally in combination with cryosurgery. When peplomycin was administered 5 min, 1 hr, and 3 hr after cryosurgery, the drug concentration in the frozen tumor was higher than that in the untreated tumor. But when administered 1 hr before cryosurgery, peplomycin was not trapped in the tumor. Trapping of adriamycin was not observed after the same treatment. In cryochemotherapy, it is necessary to administer the appropriate drug at the appropriate time. However, the trapping of the anticancer drug results in a high concentration and lasts for a long time, so that cryochemotherapy is expected to be a new mode of cancer therapy, particularly as a multidisciplinary treatment for cancer.     

Antitumor immunologic reactivity in the relatively early period after cryosurgery: Experimental studies in the rat

An experimental investigation was performed on antitumor immunity in the relatively early postoperative period after cryosurgery, using a metastasizing rat's mammary tumor, MRMT-1. Two weeks after its inoculation, surgical excision of the tumor, cryosurgery, surgical excision plus inoculation with freezing-thawing produced vaccine, or surgical excision plus fasting for 72 hr was performed, and postoperative follow-up was done on incidences of metastases, those of metastatic death, etc. Specific immunologic reactivity was examined in the surgical excision (SE) and cryosurgery (CR) groups.The FTV and fasting groups showed more metastatic deaths as compared with the SE group. The CR and SE groups did not differ significantly from each other in incidences of lung and lymph node metastases.Specific footpad reactivity at 2 and 3 weeks after treatment was lower in the CR group than in the SE group.Winn's neutralization assay showed an inhibition of tumor growth at 1 and 3 week(s) after treatment both in the SE and in the CR groups, the inhibitory effect tending to be lower in the latter.Inactivated serum obtained at 1 week after treatment showed a facilitation of tumor growth in the SE group and a tendency of tumor suppression in the CR group, showing a significant difference between them.A mild reduction in antitumor immunity seen in the relatively early postoperative period following cryosurgery probably was not due to a blocking effect by superfluous antigens. Rather it was considered to be due to activation of suppressor cells, consequent on cryosurgical stress, and/or slow and steady absorption of antigens.     

Cryosurgery and rhTNF-α play synergistic effects on a rat cortex C6 glioma model

Glioma, a type of brain tumor originating from glioma cells, varies widely in aggressiveness and causes serious symptoms, but the treatments are limited. Studies have shown that cryosurgery has multiple effects on tumor treatments, and administration of human tumor necrosis factor-alpha (rhTNF-α) arguments the anti-tumor effect of cryotherapy in breast and prostate cancers. To test the hypothesis that cryosurgery and rhTNF-α play synergistic effects against brain tumors, we established a brain glioma model on rat cortex regions following different treatments: the G1 group was sham-operated; the G2 group was treated with cryosurgery; the G3 group was treated with rhTNF-α; and G4 group received combined treatment with cryosurgery and rhTNF-α. Tumor sizes were measured by magnetic resonance imaging; DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay); P21(WAF1/CIP1) and proliferating cell nuclear antigen (PCNA) expression levels were scored using immunohistochemical staining. G2 and G4 rats had significantly longer survival time than did G1 rats. Tumor sizes in each group were significantly decreased as compared with those in G1 rats. PCNA-positive cells were significantly decreased in G2, G3 and G4 rats as compared with G1 rats. In contrast, DNA fragmentation and P21(WAF1/CIP1)-positive cells were significantly increased in each treatment group. Importantly, a combined treatment enhanced the effects of cryosurgery. Combined treatment with cryosurgery and rhTNF-α may have a synergistic effect on glioma tumor therapy, enhancing the inhibition of proliferation and the induction of apoptosis.     

The ability of induced osteo-progenitor cells to maintain and rebuild long-term ectopic osteo-hematopoietic foci in vitro

Tooth matrix particles were implanted s.c. into mice. In six to nine months, fully developed, induced osteo-hematopoietic foci complete with a cortex and central marrow cavity were formed. The cells from the induced foci were serially retransplanted under the renal capsule of syngeneic recipients. The focus formation was observed during three consecutive passages. The effect was not due to secondary induction. Our data show that the induced osteogenic cells are capable of long-term maintenance, remodeling and recurrent rebuilding of hematopoietic stroma without an additional application of the inductor.     

Cryosurgery of normal and tumor tissue in the dorsal skin flap chamber: Part II--injury response

It has been hypothesized that vascular injury may be an important mechanism of cryosurgical destruction in addition to direct cellular destruction. In this study we report correlation of tissue and vascular injury after cryosurgery to the temperature history during cryosurgery in an in vivo microvascular preparation. The dorsal skin flap chamber implanted in the Copenhagen rat, was chosen as the cryosurgical model. Cryosurgery was performed in the chamber on either normal skin or tumor tissue propagated from an AT-1 Dunning rat prostate tumor, as described in a companion paper (Hoffmann and Bischof, 2001). The vasculature was then viewed at 3 and 7 days after cryoinjury under brightfield and FITC-labeled dextran contrast enhancement to assess the vascular injury. The results showed that there was complete destruction of the vasculature in the center of the lesion and a gradual return to normal patency moving radially outward. Histologic examination showed a band of inflammation near the edge of a large necrotic region at both 3 and 7 days after cryosurgery. The area of vascular injury observed with FITC-labeled dextran quantitatively corresponded to the area of necrosis observed in histologic section, and the size of the lesion for tumor and normal tissue was similar at 3 days post cryosurgery. At 7 days after cryosurgery, the lesion was smaller for both tissues, with the normal tissue lesion being much smaller than the tumor tissue lesion. A comparison of experimental injury data to the thermal model validated in a companion paper (Hoffmann and Bischof 2001) suggested that the minimum temperature required for causing necrosis was -15.6 +/- 4.3 degrees C in tumor tissue and -19.0 +/- 4.4 degrees C in normal tissue. The other thermal parameters manifested at the edge of the lesion included a cooling rate of approximately 28 degrees C/min, 0 hold time, and a approximately 9 degrees C/min thawing rate. The conditions at the edge of the lesion are much less severe than the thermal conditions required for direct cellular destruction of AT-1 cells and tissues in vitro. These results are consistent with the hypothesis that vascular-mediated injury is responsible for the majority of injury at the edge of the frozen region in microvascular perfused tissue.     

Analysis of thermal stress in cryosurgery of kidneys

In this study, the thermal stress distribution in cryosurgery of kidney was investigated using a multiphysics finite element model developed in ANSYS (V8.1). The thermal portion of the model was verified using experimental data and the mechanics portion of the model (elastic) was verified using classic analytical solutions. Temperature dependent thermal and mechanical properties were used in the model. Moreover, the model accounts for thermal expansion due to both thermal expansion in single phase and volumetric expansion associated with phase change of tissue water to ice. For a clinical cylindrical cryoprobe inserted into the renal cortex from the top-middle renal capsule, it was found that the thermal stress distributions along the radial position are very different at different depths from the top renal capsule. The thermal stress is much higher at both ends than in the middle of the cryoprobe surface. It was found that there might be more tissue next to the top renal capsule than other region undergoing microcrack formation or plastic deformation. Furthermore, it was found that macrocrack formation is more likely to occur in tissue adjacent to the cryoprobe surface (especially on the sharp point tip) and during the thawing phase of cryosurgery. It was further found that the volumetric expansion associated with phase change induced much higher thermal stress than thermal expansion in a single phase and might therefore be the main cause of the frequently observed crack formation shortly after initiation of thawing in cryosurgery. Because the thermal stress adjacent to the cryoprobe is much higher than the yield stress of frozen renal tissue, a plastic stress model is required for better modeling of the thermal stress distribution in cryosurgery of kidney in future. However the computational effort will then be drastically increased due to the strong nonlinear nature of the plastic model and more experimental studies are indispensable for better understanding of the mechanical behavior of frozen tissue in cryosurgery.     

Cellular specificity of the cure for neonatal osteopetrosis in the ia rat

Osteopetrosis in the ia/ia rat is known to be the result of reduced bone resorption due to abnormal osteoclasts. Studies in this mutant have shown that mononuclear cells from normal littermates could cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia/ia rats. This investigation was pursued in an attempt to determine the cellular source of this cure by transplanting various populations of cells from 21-day-old normal rats to unrelated newborn ia/ia recipients. The effects of treatment were evaluated radiographically and by measuring the size of the tibial marrow cavity. The cellular suspensions that were effective in curing the disease were the Ficoll-Hypaque isolate of spleen, bone marrow, and newborn livers. The Ficoll-Hypaque isolates of lymph node, thymus, and blood, and the adherent pool of peritoneal cells and splenic cells did not produce a cure in the ia/ia recipients. These results suggest that the cellular source of the cure is a stem cell. This conclusion is further substantiated by the finding that Thy 1.1 antigen (a stem cell marker in the rat) is expressed on a majority of the cells from the donor sources that affected a cure.     

Transplantation of murine bone marrow without prior host irradiation.

The experiments presented test the hypothesis that pluripotential stem cells (assayed in the mouse as CFU-S) are normally not in cycle and that the failure of normal marrow transfusions to take in normal recipients is due to the absence of a stimulus to turn CFU-S into cycle. Following marrow transfusion from male donors into female isogeneic recipients, spleen, liver, and various parts of the skeleton were shielded to protect transfused donor cells from lethal doses of radiation gives to the rest of the body. Percentages of hemopoietic donor and host cells were subsequently determined by karyotyping C banded marrow and spleen metaphases and identifying of Y chromosome. The results support the notion that the failure of normal marrow to take in normal recipients is not due to inadequate numbers of transfused cells. Permanent colonization by donor cells, however, requires not only triggering CFU-S into cycle, but also emptying of 'niches' normally occupied by endogenous CFU-S. Partial body radiation meets both requirements. In addition, the results indicate that recently arrived donor cells, protected in the shielded portion of the body, seed more readily into the irradiated areas of the skeleton than do similarly protected host cells.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. II. Antigen-dependent enhancement by exogenous prostaglandins of the E series.

The spleen cells from CFW/D mice injected with dimethylbenzanthracene-induced leukemia virus exhibited a progressive decline in the in vitro response to heterologous erythrocyte antigens in parallel with tumor growth. Cell transfer experiments revealed that this immunodepressed state may involve a B-cell defect rather than extrinsic factors in the cellular environment since: (i) nonresponsiveness could be transferred to irradiated non-tumor-bearing mice with spleen cells, and (ii) T cells from tumorbearing mice cooperated with normal bone marrow cells, but bone marrow from tumorbearing mice did not cooperate with normal T cells. In addition, T cells from the thymic tumor could cooperate with normal bone marrow cells upon transfer to irradiated recipients. TL 485-2 cells, a T-cell line derived from the tumor, could be specifically activated with SRBC thereby indicating that the virus transformed T cells were immunocompetent. Suppressor cells, which appeared in the spleen concomitant with immunodepression and tumor development, may directly raise B-cell thresholds for T-dependent triggering signals since the antibody response of spleen cells from tumor-bearing mice could be restored by adding agents such as LPS, 2 mercaptoethanol, or T cells exogenously preactivated in normal animals. The suppressor cell could be enriched by adherence to plastic and was removed by treatment with carbonyl iron. In addition, it was unlikely that the suppressor cell was a virus-infected cell since transformed, virus-infected cells from the tumor or TL 485-2 cells were not suppressive when added to spleen cells in vitro but rather resulted in a marked, polyclonal enhancement of the PFC response. The interaction of TL 485-2 cells and normal spleen cells resulted in the release of a stimulatory factor which increased DNA synthesis in resting cells as well as increasing PFC. The role of these enhancing factors and suppressor cells in controlling tumor growth remains to be elucidated.     

Investigation of the mechanism and the effect of cryoimmunology in the Copenhagen rat

This study examined the potential for "cryoimmunology" to increase the destruction of the Dunning AT-1 prostate tumor after cryosurgery. Two possible mechanisms explaining the cryoimmunologic response were studied. The first was that an antitumor antibody is produced after cryosurgery. The second was that freezing induces an immunostimulatory signal that creates a T-cell response to the tumor. Six groups of animals (three experimental groups and three control groups) were treated once per week for 4 weeks with different therapies designed to investigate these mechanisms. Three types of immune response were measured: (1) the anti-AT-1 tumor immune titer (Ab response) by serum ELISA, (2) the effect on secondary tumor growth after challenge with live AT-1 cells (size and weight of the secondary tumor over time), and (3) the nature of the immunologic infiltrate into the secondary tumors by immunoperoxidase stain. ELISA showed that immune titers were present in the experimental groups after therapy, but the presence of an immune titer did not have a significant effect on tumor propagation. Histology showed the immunologic infiltrate was similar in all groups. These results showed that an immune response to AT-1 tumor was measurable by serum antibody, but it did not significantly limit secondary tumor growth or affect tumor histology. This suggests that the growth of AT-1 tumors is not inhibited by a cryoimmunological response. Thus, the effect of in vivo cryosurgery in the AT-1 tumor system would likely be limited to cellular and vascular changes.     

Spirogermanium: effects on hematopoietic stem cells and survival of normal and tumor-bearing mice

The effect of spirogermanium (SG) on hematopoietic stem cells, tumor burden, and survival times was investigated in C3H mice with transplanted mammary carcinoma. Compared to normal mice, the number of hematopoietic stem cells, or colony-forming units per spleen (CFU-S), was lower in the marrow of tumor-bearing mice. Spirogermanium at 15 and 30 mg/kg was not toxic to the normal hematopoietic cells in the marrow of either normal or tumor-bearing mice. In contrast to animals treated with cyclophosphamide, SG did not decrease the tumor growth rate or prolong the survival times of tumor-bearing C3H mice. Doses of 35-40 mg/kg SG did not prolong the survival times or decrease the tumor burden of AKR/J mice with a long-passaged lymphoma. These studies demonstrate that SG has minimal inhibitory effects to the marrow of normal mice and may promote the maintenance of normal marrow cells in tumor-bearing animals. However, in two different transplanted tumor cell lines, SG did not inhibit tumor growth or prolong host survival time.     

A granulocytosis-inducing tumor inhibits the production of B lymphocytes in murine bone marrow

Mice bearing a transplantable CE mammary carcinoma have been shown to have greatly augmented rates of neutrophil production coupled with a marked diminution of bone marrow lymphocytes. The objective of the present study was to test whether the loss of lymphocytes, and especially of B cells, from the bone marrow and spleen of tumor-bearing animals was due to a reduced rate of cell production and if so, at what level this response was regulated. A modified 3H-TdR pulse and chase analysis was used to assess the rates of production of small lymphocytes and B cells (stained for c mu and s mu) at weekly intervals after CE tumor transplantation. 3H-TdR was infused continuously for 24 hr, and radioautographs were prepared of bone marrow and spleen cells 0, 24, and 48 hr after termination of the infusion. Pre-B cells (c mu+s mu-) essentially disappeared from the femoral bone marrow by the end of 1 wk of tumor growth, followed by a great reduction in the number of c mu+s mu+ cells in the marrow and s mu + cells in the spleen. Although pre-B cells appeared in the peripheral marrow (caudal vertebrae, metatarsal bones) and spleen of tumor-bearing mice, these cells could not compensate for the continued decrease in the numbers of more mature B cells. In normal mice, during the 48-hr chase period, newly formed, 3H-TdR-labeled, small lymphocytes and s mu+ cells continued to emerge from the prelabeled precursor compartment at a steady rate, but after 1 wk of tumor growth, the number of small lymphocytes and s mu+ cells emerging from the precursor compartment fell steadily during the 48-hr chase period. During the second and third weeks of tumor growth, a steady state appears to have been reached in B cell production, which was at a level approximately 10 times below that of normal. Because pre-B cells are normally maintained by a less mature precursor population (2), the initial disappearance of c mu+s mu- cells suggests that the CE mammary carcinoma exerts its modulatory influence on primary B cell production by inhibiting or eliminating the cells that eventually feed into the pre-B compartment. The nature of the regulatory factors apparently secreted by the tumor and the more precise identity of the target cells are under investigation.     

The effect of 3,4-disuccinyldianhydrogalactitol, N-nitrosourea and their combination on DNA synthesis in normal and mouse P388 tumor cells

The rates of incorporation of 2-14C-thymidine into DNA of leukemia P388, bone marrow, gastrointestinal mucosa and spleen cells at various time after administration of 3,4-disuccinyldianhydrogalactitol (DisuDAG), 1-methyl-1-nitrosourea (MNU), 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea (HECNU) and their combinations at different doses to mice with leukemia P388 (solid form) were studied. DisuDAG (80 mg/kg) induced the deep and the stable inhibition in DNA synthesis of leukemia P388, bone marrow and spleen cells. The combination of DisuDAG and HECNU at small doses induced the deep and the stable suppression of DNA synthesis in tumor cells, however DNA synthesis in normal dividing cells was shown to recover more rapidly than in leukemia P388 cells. Administration of the combination of DisuDAG with MNU to tumor-bearing mice induced more stable inhibition of DNA synthesis in tumor cells in comparison with MNU and DisuDAG. In vivo inhibition of DNA synthesis in leukemia P388 cells with DisuDAG and HECNU was not due to damage in pool of precursors (TCA soluble fraction).     

Hair follicle destruction and regeneration in guinea pig skin after cutaneous freeze injury

We have investigated the histological changes in hair follicles in guinea pig skin after standardized moderate and severe cryosurgery injuries. Hair follicles were permanently destroyed by cryosurgery, but more than one mechanism may be operative during follicle destruction and shedding. The mechanism depends upon the severity of the freeze. After a light freeze injury, the changes are predominantly within the hair follicle. The hair is shed at the surface and there is selective autolysis of follicular cells, but dermal connective tissue is preserved and there is little surrounding damage. However, after a severe cryoinjury as used in "tumor doses," there is destruction of dermal connective tissue and dermal scarring. The necrotic dermis is shed, taking with it the dead follicles and morphologically normal elastic tissue.     

Sensitivity of pigmented mucosa and skin to freezing injury.

Pigmented areas of canine skin and oral mucosa were subjected to freezing in situ at various temperatures for the purpose of investigating possible differences in the sensitivity of epidermal cells to cold injury. Destruction of melanocytes occurred in the range of ?4 to ?7 °C, while squamous cells resisted injury even at ?20 °C. Replacement of the lost pigmented cells occurred from the normal tissue at the periphery of the injured area. The experiments suggest that selective destruction of pigmented lesions in clinical conditions may be achieved by freezing tissue to ?4 to ?7 °C or colder, but not to exceed ?20 °C in order to avoid destruction of squamous cells. The experiments also support wider use of cryosurgery for pigmented lesions of the skin and oral cavity.     

SELF-RENEWAL OF COLONY FORMING UNITS (CFU) IN SERIAL BONE MARROW TRANSPLANTATION EXPERIMENTS

The capacity of stem cells (CFU) for self-renewal was tested by transplanting normal bone marrow (primary transplantation) and bone marrow which had been subjected to one or two earlier transplantations (secondary and tertiary transplantation) into lethally irradiated syngeneic recipients. It was found that the capacity for self-renewal is diminished within the first weeks after one or more previous transplantations. This ability of stem cells recovered after a longer interval after the previous transplantation. The time required for this recovery depended upon the number of previous transplantations and amounted to more than 1 or 2 months after one or two transplantations respectively. Shortly after transplantation the CFU/nucleated cell ratio in bone marrow was below normal and its decrease was more pronounced when the bone marrow had been transplanted more often. An increase of the ratio towards normal values was observed in the course of one month after the last transplantation. Measurements of the spleen colony size after transplantation of normal and re-transplanted bone marrow indicated that CFUs from re-transplanted marrow gave slightly smaller spleen colonies than those of normal marrow.
It is concluded that the decreased self-renewal of stem cells shortly after previous transplantations is probably not due to a limitation in the number of normal mitoses they can perform, but to a loss of stem cells by transfer to the compartment of differentiating cells.     

Developmental abnormalities of terminal deoxynucleotidyl transferase positive bone marrow cells and thymocytes in New Zealand mice: effects of prostaglandin E1

The enzyme TdT was used as a marker with which to study the ontogeny of primitive lymphopoietic cells in NZ strain mice. A marked accumulation of abnormally large, rapidly proliferating TdT+ cells was seen in the subcapsular region of the thymus cortex in the NZB and NZB/W mice. This abnormal accumulation of TdT+ thymocytes was most pronounced in the NZB/W hybrid and persisted for at least the first 16 wk of life. In addition, significantly elevated percentages of TdT+ bone marrow cells (presumptive prothymocytes) were present in NZB, NZW, and NZB/W mice between 1 and 4 wk of age, with the highest mean peak levels occurring in the NZB strain. Treatment of both normal and adrenalectomized BALB/c and NZB/W mice with pharmacologic doses (7 to 10 mg/kg) of PGE1 caused a marked, dose-dependent decrease in thymus weight and thymus cell number within 12 to 18 hr. Histologic and cell separation studies showed that this was due to the selective depletion of PNA+ TdT+ cortical thymocytes. Similarly, PGE1 caused a reversible, dose-dependent decrease in the percentage of TdT+ bone marrow cells. In contrast, PGF2 alpha, which is not therapeutically active against autoimmunity in NZB/W mice, had no detectable effect on TdT+ bone marrow cells or thymocytes in BALB/c or NZB/W mice. These results directly document the existence of abnormalities in the development of lymphopoietic precursor cells in the bone marrow and thymus cortex of NZ strain mice prior to the onset of autoimmune phenomena. The results also raise the possibility that the therapeutic efficacy of exogenous PGE1 in autoimmune NZ strain mice may be related, at least in part, to its ability to rectify the abnormal development of these early lymphoid cells.     

Defective osteoclast differentiation and function in the osteopetrotic (os) rabbit

We tested the ability of normal osteoclast progenitors found in neonatal liver and bone marrow to develop into functional osteoclasts when co-cultured with metatarsals from newborn osteopetrotic rabbits; the latter inherit an osteoclast incompetence resistant to cure by bone marrow transplantation. This system, developed by Burger and colleagues, has been shown to produce normal, functional osteoclasts when used with normal metatarsals. Our study tested the competence of the mutant skeletal microenvironment for differentiation of normal osteoclasts. Mutant and normal metatarsals were cultured alone or with normal liver, spleen, or bone marrow for up to 14 days. All normal cultures possessed a marrow cavity and contained numerous osteoclasts with cytochemical characteristics (tartrate-resistant acid phosphatase) of active cells. Mutant metatarsals co-cultured with normal spleen, liver, or bone marrow failed to develop a marrow cavity (evidence in itself of reduced bone resorption) and had osteoclasts reduced in both numbers and cytochemically detectable activity. Similar metatarsal cultures of an osteopetrotic rat mutation (incisors--absent) curable by bone-marrow transplantation exhibited marrow cavity development in mutant metatarsals co-cultured with normal spleen. These data suggest that the skeletal environment of osteopetrotic rabbits contains an inhibitor or lacks a promoter of osteoclast differentiation and function.     

Oncogene cooperation in lymphocyte transformation: malignant conversion of E mu-myc transgenic pre-B cells in vitro is enhanced by v-H-ras or v-raf but not v-abl.

Although transgenic mice bearing a c-myc gene controlled by the immunoglobulin heavy-chain enhancer (E mu) eventually develop B-lymphoid tumors, B-lineage cells from preneoplastic bone marrow express the transgene but do not grow autonomously or produce tumors in mice. To determine whether other oncogenes can cooperate with myc to transform B-lineage cells, we compared the in vitro growth and tumorigenicity of normal and E mu-myc bone marrow cells infected with retroviruses bearing the v-H-ras, v-raf, or v-abl oncogene. The v-H-ras and v-raf viruses both generated a rapid polyclonal expansion of E mu-myc pre-B bone marrow cells in liquid culture and 10- to 100-fold more pre-B lymphoid colonies than normal in soft agar. The infected transgenic cells were autonomous, cloned efficiently in agar, and grew as tumors in nude mice. While many pre-B cells from normal marrow could also be induced to proliferate by the v-raf virus, these cells required a stromal feeder layer, did not clone in agar, and were not malignant. Most normal cells stimulated to grow by v-H-ras also cloned poorly in agar, and only rare cells were tumorigenic. With the v-abl virus, no more cells were transformed from E mu-myc than normal marrow and the proportion of tumorigenic pre-B clones was not elevated. These results suggest that both v-H-ras and v-raf, but apparently not v-abl, collaborate with constitutive myc expression to promote autonomous proliferation and tumorigenicity of pre-B lymphoid cells.     

Studies on the recovery from tolerance to tumor antigens

Summary The present study investigates the potential of bone marrow cells from mice tolerant to tumor antigens to repopulate tumor-specific effector T cells. C3H/He mice were inoculated i.v. with 10⁶ 10000 R X-irradiated syngeneic X5563 plasmacytoma tumor cells three times at 4-day intervals. This regimen abrogated the ability of spleen cells from these mice to develop anti-X5563 cytotoxic and in vivo protective (tumor-neutralizing) T cell-mediated immunity as induced by i.d. inoculation of viable X5563 cells followed by surgical resection of the tumor. Since such suppression was induced in a tumor-specific way, this represented a state of antitumor tolerance. When bone marrow cells from normal or X5563-tolerant mice were transferred i.v. into 950 R X-irradiated syngeneic C3H/He mice, both groups of recipient mice generated anti-X5563 tumor immunity over a similar time course and to almost the same degree. Anti-X5563 tumor immunity induced in (C3H/He×C57BL/6) F1 mice which had been transferred with bone marrow cells from normal or X5563-tolerant C3H/He mice were mediated by T cells expressing the Ly phenotype of C3H/He, but not of C57BL/6, excluding the possibility that the antitumor effector cells were derived from recipient mice. It was also demonstrated that C3H/He mice which had been reconstituted with normal marrow were rendered tolerant when the tolerance regimen was started 7 weeks, but not 1 week after the bone marrow reconstitution. These results indicate that bone marrow cells from antitumor tolerant mice are not rendered tolerant to the tumor but can provide the potential to repopulate antitumor CTL and in vivo protective effector T cells.This work was supported by the Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan Abbreviations used: MHC, major histocompatibility complex; CTL, cytotoxic T lymphocytes; TNP, trinitrophenyl; C, complement; TNBS; trinitrobenzene sulfonate; MMC, mitomycin C