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Reproducible multi-stage progression to invasive squamous carcinoma of the epidermis has been achieved in transgenic mice expressing the HPV16 early-region genes, including the E6/E7 oncogenes, under the control of the human keratin-14 promoter/enhancer. Although 100% of K14-HPV16 transgenic animals develop hyperplastic and/or dysplastic lesions in several inbred backgrounds, including C57BL/6, BALB/c, and SSIN/SENCAR, only mice backcrossed into the FVB/n background progress to malignant squamous cell carcinomas of two pathological grades, well differentiated and moderate/poorly differentiated (WDSC or MPDSC, respectively), each displaying characteristic patterns of malignant behavior. WDSCs typically arise within the epidermis of the ear and invade deeply into the underlying dermis but fail to metastasize, whereas MPDSCs develop on the chest and truncal skin and invariably metastasize to regional lymph nodes. The transition to the malignant state, in 21% of FVB/n transgenic mice, is characterized by alteration of the repertoire of keratin intermediate filament proteins expressed within neoplastic epidermis, such that WDSCs maintain expression of keratins common to terminally differentiating stratified keratinocytes (K10), whereas MPDSCs are distinguished from WDSCs by activation of embryonic and mucosal keratins (K13, K8, and K19). Precursor hyperplastic and dysplastic lesions are characterized by a progressively increased proliferative index, striking morphological alterations in keratinocyte cell-cell and cell-matrix interactions, and extensive remodeling of the underlying dermal stroma. Remarkably, this extensive stromal remodeling, which may facilitate both angiogenesis and eventual tumor cell invasion, develops early at the dysplastic stage in all animals well before malignant conversion.

Upregulation of acidic and basic fibroblast growth factors (FGF-1 and -2), and their cognate receptors FGFR-1 and -2, has been demonstrated in a variety of epithelial malignancies. However, the patterns of FGF/FGFR expression at specific stages of epithelial carcinogenesis have not been extensively characterized. In this report, the levels of FGF-1, FGF-2, FGF-7 mRNA and their receptors FGFR-1 and FGFR-2, were investigated during epidermal carcinogenesis in transgenic mice expressing the early region of the 'high risk' papillomavirus type 16 (HPV16) under control of the human keratin-14 enhancer/promoter (K14-HPV16 transgenic mice). FGF-1 was first upregulated in dysplasias, while FGF-2 was constitutively expressed in non-transgenic, neoplastic, and malignant keratinocytes throughout carcinogenesis. Expression of FGF-7 was undetectable in non-transgenic epidermis, and remained at threshold levels at all stages of progression. In well differentiated squamous cancers, FGFR-1 was upregulated and co-localized with angiogenic capillaries in the dermis underlying dysplastic lesions and within papillary fronds of invasive cancers. In contrast, FGFR-1 was upregulated specifically within the malignant squamous cells of moderate-poorly differentiated squamous cancers. The expression of FGFR-2 was essentially constitutive in both non-transgenic and neoplastic epidermis. Collectively the data suggest that the FGF/FGFR signaling pathways may potentially contribute to several facets of multi-stage epithelial carcinogenesis, including auto- or paracrine growth stimulation, upregulation of angiogenesis, and stromal remodeling.

High-risk human papillomaviruses (HPVs), including type 16, have been identified as factors in cervical carcinogenesis. However, the presence and expression of the virus per se appear to be insufficient for carcinogenesis. Rather, cofactors most likely are necessary in addition to viral gene expression to initiate neoplasia. One candidate cofactor is prolonged exposure to sex hormones. To examine the possible effects of estrogen on HPV-associated neoplasia, we treated transgenic mice expressing the oncogenes of HPV16 under control of the human keratin-14 promoter (K14-HPV16 transgenic mice) and nontransgenic control mice with slow release pellets of 17beta-estradiol. Squamous carcinomas developed in a multistage pathway exclusively in the vagina and cervix of K14-HPV16 transgenic mice. Estrogen-induced carcinogenesis was accompanied by an incremental increase in the incidence and distribution of proliferating cells solely within the cervical and vaginal squamous epithelium of K14-HPV16 mice. Expression of the HPV transgenes in untreated transgenic mice was detectable only during estrus; estrogen treatment resulted in transgene expression that was persistent but not further upregulated, remaining at low levels at all stages of carcinogenesis. The data demonstrate a novel mechanism of synergistic cooperation between chronic estrogen exposure and the oncogenes of HPV16 that coordinates squamous carcinogenesis in the female reproductive tract of K14-HPV16 transgenic mice.

c-Myc and Mad each form heterodimers with Max that bind the same E-box related DNA sequences. Whereas Myc:Max complexes activate transcription and promote cell proliferation and transformation, Mad:Max complexes repress transcription and block c-Myc-mediated cell transformation. Here we examine these antagonistic transcription factors during epithelial differentiation and neoplastic progression. During differentiation of primary human keratinocytes, Mad is rapidly induced and c-Myc is downregulated, resulting in a switch from c-Myc:Max to Mad:Max heterodimers. In normal epidermis and colonic mucosa c-myc expression is restricted to proliferating cell layers, while mad expression is restricted to differentiating cell layers. Using HPV18 transformed keratinocytes that vary in their ability to differentiate in organotypic cultures, we find that Mad induction occurs only in those cells that retain a differentiation response. In the epidermis of transgenic mice in which expression of the HPV16 E6 and E7 oncogenes are targeted to basal keratinocytes, neoplastic progression occurs and is marked by an expansion of c-myc expressing basal-like cells. Expression of mad is found only in growth-arrested differentiating cells on the outer edges of preneoplastic lesions. The squamous cell carcinomas that arise evidence a variable number of sites within the tumor masses where mad expression and morphological differentiation coincide; increasing malignancy correlates with loss of both mad and capability to differentiate. These results indicate that c-Myc and Mad expression are tightly coupled to the transition from proliferation to differentiation of epithelial cells and that restriction of Mad expression may be associated with loss of normal differentiation capability and with tumorigenesis.

Mice transgenic for SV40 T antigen (Tag) under control of the rat insulin promoter (RIP) develop two alternative immunological phenotypes: tolerance or autoimmunity towards Tag. We utilized the T cell receptor (TCR) genes expressed in a Tag-specific CD4+ cell from an autoimmune RIP-Tag mouse to generate two lines of TCR transgenic mice in which either 10% or 90% of peripheral T cells express the transgenic TCR. When cross-bred to the tolerant RIP1-Tag2 line, mice from the low frequency TCR line showed partial deletion of peripheral Tag-specific T cells and nonresponsiveness of those that remained. In contrast, crossbred mice in which transgenic T cells comprised a majority of the T cell population were nontolerant both in vivo and in vitro. Thus, tolerization of CD4+ T cells specific for a rare self-antigen may fail if too many autoreactive T cells develop.

To model human papillomavirus-induced neoplastic progression, expression of the early region of human papillomavirus type 16 (HPV16) was targeted to the basal cells of the squamous epithelium in transgenic mice, using a human keratin 14 (K14) enhancer/promoter. Twenty-one transgenic founder mice were produced, and eight lines carrying either wild-type or mutant HPV16 early regions that did not express the E1 or E2 genes were established. As is characteristic of human cancers, the E6 and E7 genes remained intact in these mutants. The absence of E1 or E2 function did not influence the severity of the phenotype that eventually developed in the transgenic mice. Hyperplasia, papillomatosis, and dysplasia appeared at multiple epidermal and squamous mucosal sites, including ear and truncal skin, face, snout and eyelids, and anus. The ears were the most consistently affected site, with pathology being present in all lines with 100% penetrance. This phenotype also progressed through discernible stages. An initial mild hyperplasia was followed by hyperplasia, which further progressed to dysplasia and papillomatosis. During histopathological progression, there was an incremental increase in cellular DNA synthesis, determined by 5-bromo-2'-deoxyuridine incorporation, and a profound perturbation in keratinocyte terminal differentiation, as revealed by immunohistochemistry to K5, K14, and K10 and filaggrin. These K14-HPV16 transgenic mice present an opportunity to study the role of the HPV16 oncogenes in the neoplastic progression of squamous epithelium and provide a model with which to identify genetic and epigenetic factors necessary for carcinogenesis.

The effect of the human papillomavirus (HPV) oncogenes E6 and E7 was examined in transgenic mice with a construct containing the human beta-actin promoter regulating HPV16 E6 and E7 open reading frames. In the sole line of mice that transmitted the transgene, neuroepithelial tumors appeared at 2.5 months of life, and by 10 months, 87 of 122 (71%) of the animals were dead from brain tumors. The most frequent type of tumor (74%) was an anaplastic neuroepithelial tumor associated with the ependyma of the third and fourth ventricles, which locally invaded adjacent brain tissue and spread for considerable distances along the ventricular surface. The other two types of tumor were well-differentiated choroid plexus carcinomas (26%) and rare pituitary carcinomas (8.7%). HPV16 E6 RNA and E7 oncoprotein expression were demonstrated in tumor tissue and primary cell lines derived from the tumors. Examination of two tumor suppressor gene products, the retinoblastoma protein and p53, known to bind to HPV16 E7 and E6 oncoproteins, respectively, showed both were expressed in the primary tumor cell lines. These data support a causative role for the HPV oncoproteins in epithelial carcinogenesis.

The purpose of this study was to investigate whether substrate deprivation acutely and selectively decreases ATP concentration in an experimental sarcoma. Two methods of substrate deprivation were examined: glycolysis was inhibited using 2-deoxyglucose (2DG), and plasma substrate levels were reduced using insulin. The effects of treatment on tumor ATP, inorganic phosphate, and pH were studied by 31P nuclear magnetic resonance spectroscopy. 2DG (2 g/kg) was administered i.p. to rats bearing s.c. methylcholanthrene-induced sarcomas. Inhibition of glycolysis by 2DG caused a 52 +/- 13% (SE) decrease in the tumor ATP to inorganic phosphate ratio, associated with a decrease in pH of 0.38 +/- 0.10 unit. The same dose of 2DG caused no significant change in the ratio of phosphocreatine to ATP in brain. Insulin (125 units/kg, i.p.) caused a 68% decline in plasma glucose and a 71% decline in betahydroxybutyrate compared to saline-treated animals. Concomitantly, 31P nuclear magnetic resonance spectroscopy detected a 48 +/- 13% decrease in sarcoma ATP, with a reciprocal elevation of inorganic phosphate in insulin-treated animals. In contrast, the brain phosphocratine/ATP ratio was unaffected by insulin. These results suggest that large tumors are acutely sensitive to inhibition of glycolysis and reductions in plasma levels of substrates for oxidative phosphorylation and glycolysis, while the brain is unaffected. In addition, this work provides support for the use of 31P nuclear magnetic resonance spectroscopy to monitor tumor response to therapy.

Interactions between molecules control intra- and intercellular physiology. Cancer is emerging as a disease in which individual molecules are either overproduced, mutated, expressed at inappropriate stages of development, or lost due to inheritance or aberrant mitotic division. The major players in this contest of cellular control are growth factors, growth factor receptors (GFRs), signal transducers, and dominant or suppressor/recessive oncogenes. The tumors most frequently removed by surgeons have been reported to have changes in one or another of these types of molecules. The concept of multistage carcinogenesis, whereby malignancy arises after a sequence of changes that are cumulative, and passed from progenitor to daughter cells, is also being defined as a sequence of molecular, genetic, and chromosomal alterations. Molecular antineoplastic therapy is in early stages of development at the laboratory bench. The future may see patients screened for cancer susceptibility, evaluated for adjuvant therapy, and chosen for particular treatment based on molecular analysis. The types of cancer operations and the scope of surgical resection may change as molecular techniques enhance oncologic treatment.

Resting energy expenditure (REE), nutritional parameters, and substrate levels were measured using a technique to minimize artifacts in 11 control patients, nine patients with localized, and four patients with diffuse neoplastic disease. Patients with diffuse disease had significantly increased percentage preillness weight loss (15.4 +/- 7.0%), decreased arm muscle circumference (21.9 +/- 2.1 vs. 26.2 +/- 3.5 cm), serum albumin (3.6 +/- 0.6 vs. 4.4 +/- 0.2 g/dl), total lymphocyte count (1024 +/- 613 vs. 1796 +/- 495 cells/mm3), and creatinine-height index (0.68 +/- 0.16 vs. 1.18 +/- 0.37) compared to controls. Both groups of cancer patients had significantly increased REE compared to controls: 25.6 +/- 3.7 (diffuse), 21.4 +/- 3.7 (localized), vs. 18.1 +/- 2.9 kcal/kg/d (controls). However, when REE was expressed as a function of metabolic body size, the significant difference persisted only in the patients with diffuse disease compared to controls: 71.8 +/- 16.4 vs. 53.9 +/- 8.1 kcal/kg3/4/d. Patients studied pre- and posttumor resection all had a postoperative drop in their REE, which was significantly correlated with the measured tumor volume. In this homogeneous, select group of patients, the tumor-bearing state exerts a moderate impact on nutritional and metabolic parameters, which are probably related to the extent of disease.

A 52-year-old female presented with an extensive retroperitoneal sarcoma and intraluminal inferior vena caval extension. Presumably during resection, embolization of a tumor fragment occurred to the right pulmonary hilum. The patient was asymptomatic for 16 months until a right hilar mass developed on a follow-up chest x-ray. Pathological examination of the gross and microscopic sections of the pneumonectomy specimen confirmed the intra-arterial location of the tumor mass. Widespread systemic metastasis became evident soon after pneumonectomy.

The incidence and severity of wound complications were examined in 105 patients with extremity and superficial truncal sarcomas who were eligible for wide local excision with or without adjuvant perioperative brachytherapy (BRT) and/or chemotherapy. Fifty-four cases from the eligible group were entered into a randomized prospective trial of the efficacy of BRT in decreasing local recurrence. In the eligible patients, major wound complications occurred in nine of 41 (22%) of the BRT cases, compared with two of 64 (3%) of the non-BRT patients, which was a significant increase (P = .002). The combined frequency of major and moderate wound complications was also significantly increased in the BRT (18 of 41, 44%) compared with the non-BRT (nine of 64, 14%) patients (P = .0006). The median duration to complete resolution of these complications was 189 days (14 to 597) in the BRT, compared with 49 (11 to 170) days in the non-BRT group (P = .0005); however, no amputations were required, and only 14% of the BRT-associated wound complications were of prolonged duration, ie, greater than 200 days. In the randomized study, both the total number of complications, and the combination of major and moderate complications were increased significantly in the BRT v the non-BRT patients. Adjuvant Adriamycin (Adria Laboratories, Columbus, OH) administered in 60 mg/m2 increments to a cumulative dose of 540 mg/m2 did not appear to impair wound healing even when administered within 15 days of operation. Significant wound complications occur in major resections of extremity and superficial truncal sarcomas. If the addition of adjuvant BRT produces a decrease in local recurrence, then either patient selection will have to be more rigidly applied, especially in wounds where skin flap blood supply is tenuous, or the technique will need to be modified to balance the short-term aim of reducing wound complications with the long-term goal of local tumor control.

Glucose and alanine metabolism were investigated in non-tumor-bearing (NTB) and tumor-bearing (TB) male F344 rats after a 24-hr fast and during the infusion of either 0.9% NaCl solution or glucose at 0.67 or 2.35 mg per 100 g total body weight per min. During 0.9% NaCl solution infusion, the plasma glucose level was higher (98.2 +/- 4.0 versus 85.8 +/- 8.1 mg per di; p less than 0.05), the whole-blood lactate level was lower (5.8 +/- 0.8 versus 8.3 +/- 1.6 mg per di; p less than 0.05), the glucose turnover rate was lower (0.72 +/- 0.04 versus 0.88 +/- 0.13 mg per 100 g total body weight per min; p less than 0.05), alanine turnover rate and the percentage of glucose derived from alanine was measured by [14C]alanine in the NTB and compared to the TB animals. In response to glucose infusions, the whole-blood lactate level rose in both groups but remained lower (7.1 +/- 0.9 versus 10.5 +/- 2.4 mg per dl at 0.67 mg per 100 g total body weight per min, p less than 0.05; 9.1 +/- 1.1 versus 19.3 +/- 5.5 mg per dl at 2.35 mg per 100 g total body weight per min, p less than 0.05; NTB versus TB) in the NTB than in the TB animals. The endogenous production rate of glucose as measured by [3H]glucose displayed a similar response to exogenous substrate in the NTB and TB animals but required a higher plasma glucose concentration to effect a similar degree of suppression in the TB group. The alanine turnover rate rose to a similar level, and the percentage of glucose derived from alanine was similarly depressed in the NTB and TB animals at each glucose infusion rate.

Deep venous thromboembolism (DVT) was studied in 44 patients with clinical Stage I, II, and III melanoma undergoing staging and therapeutic inguinal lymph node dissection. The ability of two noninvasive methods of surveillance, the phleborheograph (PRG) and the 125I fibrinogen scan to detect deep venous thrombosis was determined by comparison with prospective bilateral lower extremity venograms. In addition, the therapeutic impact, both beneficial and detrimental, of low dose heparin, 5000 units administered subcutaneously two hours prior to and every eight hours after operation was determined in a double blind study. The sensitivity of the PRG and 125I fibrinogen scan were both 20%. There were five deep venous thrombi, and two pulmonary emboli for a combined incidence of DVT of 13.6% for the entire patient population. However, there was no significant difference in the incidence of DVT between the two groups. The heparin-treated patients had an increased total volume (796 +/- 516 versus 388 +/- 208 ml; p less than 0.05), and duration of wound drainage (9 +/- 4 versus 13 +/- 6 days; p less than 0.05).

Mucoepidermoid carcinoma of the trachea is rare. Its occurence in a 14-year-old boy is reported here. This case illustrates the typical course of tracheal tumors with clinical manifestations of cough, wheezing, and hemoptysis, the intially reported normal chest roentgenogram, and the common failure to diagnose tracheal tumor for several months. Early use of tomographic studies and bronchoscopic examination in any person with recent onset of airway obstruction unresponsive to bronchodilator therapy is recommended.

Plasma amino acid concentrations were measured in fasting nontumor bearing (NTB) and tumor bearing (TB; methylcholanthrene induced sarcoma) male Fischer F344 rats during infusion of 0.9% NaCl solution or glucose at 3.72 or 13.05 mumol/100 g total body weight (TBW)/min. The animals were studied when the tumor comprised only 8% of the TBW at a time when decreased food intake and weight loss were not manifest. During 0.9% NaCl infusion there were no significant differences between NTB or TB animals in the concentration of alanine (NTB: 152.6 +/- 20.1; TB: 150.3 +/- 19.0 microM; mean +/- SD), branched chain amino acids (BCAA) (NTB: 343.3 +/- 48.7; TB: 344.2 +/- 20.5 microM), essential amino acids, aromatic amino acids, or total amino acids. During infusion of glucose at 3.72 mumol/100 g TBW/min the alanine levels rose (NTB: 283.6 +/- 33.4; TB: 286.7 +/- 43.3 microM), and the BCAA levels fell (NTB: 215.9 +/- 19.4; TB: 228.7 +/- 43.4 microM) to similar concentrations in both NTB and TB animals. Glucose infusion at 13.05 mumol/100 g TBW/min resulted in an additional increase in the alanine concentration (NTB: 344.5 +/- 28.7; TB: 382.8 +/- 116.6 microM), and a further decrease in the BCAA concentration (NTB: 166.4 +/- 30.8; TB: 160.7 +/- 30.5 microM) without significant differences between NTB and TB animals. Paired analysis for each animal prior to and during glucose infusion demonstrated a similar absolute micromolar change in alanine and BCAA concentration during both glucose infusion rates in both NTB and TB animals. The levels of aromatic amino acids and total amino acids were unchanged and the essential amino acid concentrations were decreased only at the higher glucose infusion rate in both NTB and TB groups. Basal amino acid metabolism appears similar in the NTB and TB animals, prior to the onset of anorexia and weight loss. During exogenous glucose infusion the reciprocal changes in the plasma alanine and BCAA concentrations support the concept of a glucose-alanine-BCAA cycle at the whole body level that appears to respond to a similar extent in NTB and TB animals.

The purpose of this study was to determine whether the energy metabolism of an experimental rodent sarcoma was selectively depressed by the combination of inhibition of glycolysis and respiration. In vivo phosphorus-31 nuclear magnetic resonance spectroscopy was used to monitor the response of tumor or brain high-energy phosphate compounds to insulin hypoglycemia, rhodamine 123, or both agents in fasting rats with subcutaneous methylcholanthrene-induced sarcomas. Insulin or rhodamine 123 alone produced a similar 50% to 60% reduction in tumor adenosine triphosphate (ATP) concentration compared with controls injected with saline solution (p less than 0.05, one-way analysis of variance [ANOVA]). The combination of insulin plus rhodamine 123 resulted in a 90% reduction of tumor ATP concentration, which was significantly different from the effect of either agent alone (p less than 0.05, one-way ANOVA). Brain phosphocreatine and ATP concentrations were unchanged by these agents. Administration of dimethyl sulfoxide (DMSO)/glycerol, the vehicle for rhodamine, produced a 35% reduction of tumor ATP, which was similar to the effect of insulin alone but significantly different from rhodamine. The combination of DMSO/glycerol plus insulin hypoglycemia resulted in a 70% reduction in tumor ATP, which was significantly elevated compared with the combination of rhodamine plus insulin. Glucose deprivation induced by insulin, and combined with the inhibition of oxidative phosphorylation, produces an additive depression of tumor energetics. The drug vehicle DMSO/glycerol significantly depresses tumor energy metabolism, presumably because of its DMSO component, which may explain the previously reported antineoplastic efficacy of this solvent. Combinations of inhibitors directed at different points of tumor metabolism produced an enhanced depression of tumor energetics, whereas host tissue was protected.