Clinical impact of coexisting retinopathy and vascular calcification on chronic kidney disease progression and cardiovascular events.

BACKGROUND AND AIMS: Retinopathy and vascular calcification (VC) are representative markers of microvascular and macrovascular dysfunction in patients with chronic kidney disease (CKD). However, their relationship and combined effects on clinical outcomes remain undetermined. METHODS AND RESULTS: We included 523 patients with nondialysis-dependent CKD stage 3-5 who had been examined with fundus photography for diabetic or hypertensive retinopathy. Simple radiographs were analyzed for the presence of VC. The clinical significance of VC of the abdominal aorta and iliofemoral artery (apVC) and retinopathy was evaluated in terms of the rate of renal function decline and composite of any cardiovascular event or death. CKD patients with retinopathy showed higher prevalence of apVC than those without retinopathy (25.6% vs. 12.5%, P < 0.001).The presence of retinopathy was independently associated with apVC (OR 2.13, 95% CI 1.31, 3.49). In multivariate analysis, compared with subjects with neither apVC nor retinopathy, the coexistence of both apVC and retinopathy were independently associated with rapid renal function decline (ß = -1.51; 95% CI -2.40, -0.61), whereas apVC or retinopathy alone were not. Compared with subjects with neither apVC nor retinopathy, the HRs for composite end points were 1.05 (95% CI 0.48, 2.27), 1.79 (95% CI 1.14, 2.80), and 2.07 (95% CI 1.17, 3.67) for patients with apVC only, those with retinopathy only, and those with both apVC and retinopathy, respectively. CONCLUSION: The coexistence of VC and retinopathy was independently associated with CKD progression and cardiovascular events or deaths, and its combined effect was stronger than any separate condition.

Disseminated mucormycosis with myocardial involvement in a renal transplant recipient.

We report the case of a renal transplant recipient with pulmonary and splenic mucormycosis whose demise was accelerated by a myocardial abscess. Once pulmonary and splenic mucormycosis was diagnosed, liposomal amphotericin B was started and immunosuppressant treatments were discontinued. The pulmonary cavities regressed during treatment, but new myocardial and peri-allograft abscesses developed. The myocardial abscess diffusely infiltrated the left ventricular wall and was associated with akinesia, which led to sudden cardiac arrest. This case demonstrates a rare manifestation of mucormycosis and highlights the fatality and invasiveness of this infection.

High normal albuminuria is associated with arterial stiffness and carotid atherosclerosis in Korean patients with type 2 diabetes.

BACKGROUND AND AIMS: Current evidence suggests that high normal albuminuria is significantly associated with increased cardiovascular risk. This study investigated the association between urine albumin-to-creatinine ratio (ACR) within the normal range and subclinical atherosclerosis in Korean patients with type 2 diabetes. METHODS AND RESULTS: This cross-sectional study involved 521 type 2 diabetic patients with normoalbuminuria. Brachial-ankle pulse wave velocity (baPWV) measurement and ultrasound assessment of carotid atherosclerosis was done. Subclinical atherosclerosis was assessed by the presence of high baPWV (>1682 cm/s), carotid atherosclerosis (intima-media thickness >0.8 mm or the presence of plaques), and carotid stenosis (≥50% of luminal narrowing). Across the tertiles of ACR, there was a trend for an increasing prevalence of high baPWV (16.8%, 20.0%, and 38.2%, P < 0.001), carotid atherosclerosis (46.9%, 55.4%, and 64.7%, P < 0.001), and carotid stenosis (12.7%, 16.0%, and 30.1%, P < 0.001). In multivariate analysis, patients in the highest ACR tertile had an odds ratio of 2.05 (95% confidence interval [CI], 1.13-3.72, P = 0.019) for high baPWV, 1.78 (95% CI, 1.08-2.93, P = 0.024) for carotid atherosclerosis, and 2.72 (95% CI, 1.44-5.11, P = 0.002) for carotid stenosis compared to those in the lowest tertile. The relation of ACR with carotid atherosclerosis and stenosis remained significant even in patients without diabetic retinopathy. CONCLUSION: High normal albuminuria was significantly associated with atherosclerotic vascular changes, independent of retinopathy and other cardiovascular risk factors. High normal albuminuria may be an early marker for subclinical atherosclerosis in type 2 diabetes.

Characterization of NODs and TLRs in innate immune response of human cementoblast cells.

OBJECTIVES: Microbial Pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and the nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. In this study, we examined the characterization of NODs and TLRs on innate immune responses in human cementoblast (HCEM) cells. MATERIALS AND METHODS: The gene expression of NODs and TLRs was examined by RT-PCR. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) productions in culture supernatants were measured by ELISA. Western blot analysis was performed to determine the degradation of IκB-α and Mitogen activated protein kinase (MAPK) activation in response to their agonist. RESULTS: The levels of NODs and TLRs were apparently expressed in HCEM cells. Although a few gene levels were weak in intact cells, the stimulation by their agonists increased the gene expression of TLRs. NODs and TLRs led to the production of IL-6 or IL-8 and the degradation of IκB-α and MAPK activation in HCEM cells. Combination treatment of NOD1 or NOD2 agonists with TLRs agonists did not influence the production of IL-6 and IL-8 in HCEM cells. CONCLUSIONS: Our results indicate that NODs and TLRs are functionally expressed in HCEM cells and can trigger innate immune responses. However, NOD1 and NOD2 may not be cooperated with TLRs to elicit an immune response in HCEM cells.

Long-term risk of hypertension and chronic kidney disease in living kidney donors.

OBJECTIVE: The aim of this study was to assess the long-term risks of chronic kidney disease and arterial hypertension in living kidney donors. METHODS: Donors who were followed for more than 1 year after nephrectomy were included. We assessed each donor's blood pressure, urine protein, and estimated glomerular filtration rate (eGFR). RESULTS: The follow-up rate was 11% (154 out of 1,356 donors), only 19% of whom were followed by nephrologists. Blood pressure had increased from 113/75 to 116/77 mm Hg (P < .01), urinary protein excretion after donation did not increase, and renal function was well preserved after donor nephrectomy. However, 33 patients (21.4%) showed a decreased eGFR of <60 mL/min/1.73 m(2), and 3 donors developed end-stage renal disease that required renal replacement therapy. CONCLUSIONS: The follow-up rate of living donors after donation was low, and we observed an increased risk of developing chronic kidney disease after donation.

Safety and efficacy of a quinolone-based regimen for treatment of tuberculosis in renal transplant recipients.

BACKGROUND: Rifampin (RFP) is a first-line antituberculosis drug, but it increases the risk of acute rejection (AR) in transplant recipients. This study evaluated whether quinolone (QNL) can replace RFP in renal transplant recipients with tuberculosis. METHODS: One hundred nine patients with active tuberculosis were included. Patients consisted of RFP (n = 91) and QNL (n = 18) groups based on the initial treatment regimen. Patients with RFP-associated adverse effects were subdivided into RFP-maintenance (RFP-M; n = 18) and QNL-conversion (QNL-C; n = 8) groups. Clinical outcomes were compared between groups. RESULTS: The incidence of AR was higher in the RFP group than in the QNL group (24.2% vs 5.6%). The QNL group showed significantly higher 10-year graft survival rates than the RFP group (88.1% vs 66.5%; P = .022). The QNL-C group showed significantly higher 10-year graft survival rates than the RFP-M group (87.5% vs 27.8%; P = .011). The rate of complete functional recovery after AR was higher in the QNL-C group than in the RFP-M group (50% vs 22.2%). CONCLUSIONS: A QNL-based regimen may be safe and effective for treatment of tuberculosis and may lower the risk of graft failure in renal transplant recipients.

Proyl isomerase Pin1 facilitates ubiquitin-mediated degradation of cyclin-dependent kinase 10 to induce tamoxifen resistance in breast cancer cells.

Endocrine therapies that inhibit estrogen receptor (ER)-α signaling are the most common and effective treatment for ER-α-positive breast cancer. However, the use of these agents is limited by the frequent development of resistance. The aim of this study was to elucidate the mechanisms by which downregulation of CDK10 expression confers resistance to tamoxifen in breast cancer. Here, we show that peptidyl-prolyl isomerase Pin1 downregulates CDK10 protein as a result of its interaction with and ubiquitination of CDK10, thereby affecting CDK10-dependent Raf-1 phosphorylation (S338). Pin1(-/-) mouse embryonic fibroblasts (MEFs) show higher CDK10 expression than Pin1(+/+) MEFs, whereas CDK10 protein was downregulated in the rescued Pin1(-/-) MEFs after reexpression of Pin1. Pin1 silencing in SKBR-3 and MCF7 cells increased the CDK10 expression. In human tamoxifen-resistant breast cancer and tamoxifen-resistant MCF7 cells, immunohistochemical staining and immunoblotting analysis shows an inverse correlation between the expression of CDK10 and the degree of tamoxifen resistance. There was also a positive correlation between the high level of P-Raf-1 (Ser338) and Pin1 in human tamoxifen-resistant breast cancer and tamoxifen-resistant MCF7 (TAMR-MCF7) cells. Importantly, 4-OH tamoxifen (4-OHT), when used in combination with overexpressed CDK10 or Raf-1 inhibitor, increased cleaved PARP and DNA fragmentation to inhibit cologenic growth of MCF7 cells and Tamoxifen-resistant MCF7 cells, respectively. On the basis of these findings, we suggest that the Pin1-mediated CDK10 ubiquitination is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

Influence of sirolimus on cyclosporine-induced pancreas islet dysfunction in rats.

This study was performed to investigate the effect of sirolimus (SRL) on cyclosporine (CsA)-induced pancreatic islet dysfunction in rats. Three separate studies were performed. First, diabetogenic effect of SRL was evaluated with three different doses (0.15, 0.3 and 0.6 mg/kg). Second, rats were treated with SRL (0.3 mg/kg) with or without CsA (15 mg/kg) for 4 weeks. Third, rats were treated with CsA for 4 weeks, and then switched to SRL for 4 weeks. The effect of SRL on CsA-induced pancreatic islet dysfunction was evaluated by an intraperitoneal glucose tolerance test, plasma insulin concentration, HbA1c level, HOMA-R index, immunohistochemistry of insulin and pancreatic beta islet cell mass. The SRL treatment increased blood glucose concentration in a dose-dependent manner. The combined treatment with SRL and CsA increased blood glucose concentration, Hemoglobin A1c (HbA1c) level, HOMA-R [fasting insulin (mU/mL) x fasting glucose (mmol/L)]/22.5] index and decreased plasma insulin concentration, immunoreactivity of insulin and pancreatic beta islet cell mass compared with rats treated with CsA. CsA withdrawal for 4 weeks improved pancreatic beta-cell function and structure. However, conversion from CsA to SRL further increased blood glucose levels compared with the rats converted from vehicle to SRL. The results of our study demonstrate that SRL is diabetogenic and aggravates CsA-induced pancreatic islet dysfunction.

Left-hand-assisted laparoscopic resection of hepatocellular carcinoma in an accessory liver.

We report a left-hand-assisted laparoscopic resection of hepatocellular carcinoma that developed in an accessory liver in a 47-year-old man. Preoperative assessment of the location of the tumor and the feeder vessels by combined selective angiography and computed tomography studies predicted the feasibility of laparoscopic procedures for complete removal of the tumor. In an attempt to avoid direct contact of the tumor capsule with rigid instruments during the operation, left-hand-assisted procedures were attempted. The encapsulated mass, 6 x 5 x 3 cm in size, was located on the posterior side of the left diaphragm, and a thin stalk between the tumor and the margin of the left lateral segment of the liver proper was recognized. Hand-assisted procedures ensured the complete mobilization of the lesion with an adequate margin, without any unexpected capsular tear. Left-hand-assisted laparoscopic procedures would be feasible for the easy and safe resection of localized hepatocellular carcinoma developing in an accessory liver.

Hand-assisted laparoscopic splenectomy for idiopathic thrombocytopenic purpura during pregnancy.

A successful case of a hand-assisted laparoscopic splenectomy with low-pressure pneumoperitoneum for autoimmune thrombocytopenic purpura in a patient at 23 weeks' gestation is reported. Preoperative splenic arterial embolization was performed on the same day as the operation using painless contour embolic material and super-absorbent polymer microspheres. The abdominal wall retraction method first was applied to avoid the effects of pneumoperitoneum on systemic hemodynamic alterations. However, a sufficient surgical view could not be obtained, as the intra-abdominal organs were elevated because of the enlarged uterus. A surgical view with 4 to 6-mm Hg pneumoperitoneum was available for the hand-assisted splenectomy. The postoperative course was uneventful, and the patient vaginally delivered a healthy infant. A hand-assisted laparoscopic splenectomy with low-pressure pneumoperitoneum after splenic arterial embolization would be feasible for patients with autoimmune thrombocytopenic purpura during a relatively advanced pregnancy.

Ventilatory muscle recruitment and work of breathing in patients with respiratory failure after thoracic surgery.

OBJECTIVES: Increased work of breathing (WOB) and respiratory muscle weakness have been identified as major causes of respiratory failure after thoracic surgery. This study was undertaken firstly to characterize the mechanical impairment in patients with respiratory failure after cardio-thoracic surgery, and secondly, to determine how diaphragmatic paralysis affects deterioration in the ventilatory mechanics. METHODS: We evaluated the respiratory mechanics of 24 patients following cardiac and thoracic surgery. Ten patients without respiratory problems were examined as control subjects. There were nine patients with phrenic nerve injury and five patients without phrenic nerve injury who required mechanical ventilation for more than 7 days. Phrenic nerve injury was assessed with a phrenic nerve stimulation test. We measured the respiratory variables, the esophageal, gastric and transdiaphragmatic pressure swing (deltaPes, deltaPga and deltaPdi, respectively), and the work of breathing during quiet tidal breathing. RESULTS: Both the groups requiring mechanical ventilation exhibited abnormally negative deltaPga/deltaPes values, compared with the control subjects. A significant increase in WOB with the normal generation of deltaPdi was seen in the patients without phrenic nerve injury. In contrast, the poor generation of deltaPdi with a slight increase in work of breathing was noted in patients with phrenic nerve injury. CONCLUSIONS: These results demonstrated two different types of respiratory failure in thoracic surgery patients, focusing on the impact of phrenic nerve paralysis. Diaphragmatic dysfunction should not be overlooked in postoperative care, and the amelioration of this compromise in respiratory mechanics is an important aspect of good patient management.

Clinical spectrum of congenital cystic disease of the lung in children.

OBJECTIVES: Congenital cystic lesions of the lung are uncommon but share similar embryologic and clinical characteristics. The purpose of this study is to review our institutional experience of congenital cystic lung disease, emphasizing the clinical spectrum of the disease related to age, and present some cases with unusual clinical manifestations. PATIENTS: Between 1962 and 1996, 26 patients (9 females and 17 males) under 15 years old underwent evaluation and surgical treatment for congenital cystic lung disease. Seven patients were under 1 year old, and 19 were in over 1 year old. There were 13 bronchogenic pulmonary cysts, 6 pulmonary sequestrations, 4 congenital cystic adenomatoid malformations (CCAM), and 3 congenital lobar emphysemas. RESULTS: All patients under 1 year old showed respiratory distress with mediastinal shift but no episodes of infection. In contrast, 13 of the 19 patients over 1 year old had symptoms of recurrent infection without respiratory distress. Five patients over 1 year old were entirely asymtomatic from birth. There were significant differences (P < 0.05) in the frequencies of respiratory distress and infection between the two groups (chi2-test). Lobectomy was performed in 21 patients, excision in 3 patients, segmentectomy in one patient, and exploration in one patient. There was no incident of postoperative mortality or morbidity except for one patient with CCAM complicated by reexpansion lung edema. Twenty-one patients at long-term follow-up from 2 to 30 years after surgery are doing well with no subsequent limitation of physical activities due to lung resection. CONCLUSIONS: In patients under 1 year old, cystic lesions were discovered by respiratory distress; and in patients over 1 year old signs of infection were the most important clinical features. Early recognition of these relatively rare congenital cystic lung lesions would lead to the immediate, proper surgical intervention.

[Reconstruction of great vessel for patients with advanced lung cancer or malignant mediastinal tumor].

Fifteen lung cancer (LC) and 11 malignant mediastinal tumor (MMT) patients who received surgical treatment in association with a reconstruction of great vessel from 1981 to 1997 were retrospectively reviewed. The aorta was resected in 7 (AO group) of 15 LC patients and the superior vena cava in the remaining 8 patients (SVC group). Pathological examination revealed actual tumor invasion in one (14%) of AO group and 6 (75%) of SVC group. In 7 p-T4 patients, 6 (85.7%) were p-N2 or N3 diseases. Five of the 6 patients belonged to SVC group and one to AO group. Five-year survival rate was 10% in all LC patients, 20% in p-T3 patients, 0% in p-T4 cancers, 50% in p-N0 patients and 0% in p-N2 or N3 patients. Eleven MMT patients included 5 thymomas, 2 thymic cancers and 4 germ cell tumors. SVC was resected and replaced with ringed PTFE graft in all patients. Invasion of vessel wall was diagnosed pathologically in 10 of 11 patients. Fifteen-year survival rate was 60% in 5 thymoma patients but 4 of whom had distant metastasis. Five-year survival rate was 50% in thymic cancers and 0% in germ cell tumors. Since the prognosis of LC and MMT patients who have tumor invasion of the great vessel is very poor, induction therapy based on preoperatively pathological diagnosis is recommended.

Results of surgical treatment of thymomas with special reference to the involved organs.

OBJECTIVE: The purpose of this study is to clarify the significance of the particular involved organ as a prognostic factor and its relation to other previously reported factors. METHODS: The prognoses of 194 consecutive patients with thymoma who had undergone complete or subtotal resection were reviewed retrospectively. Survival was evaluated as actuarial freedom from tumor death. Analysis of prognostic factors was performed by the Kaplan-Meier method with the log rank test and Cox's proportional hazards model. RESULTS: The Masaoka staging system and involvement of the great vessels were the independent prognostic factors in the entire study group; age, sex, histologic subtype, completeness of resection, association of myasthenia gravis, or involvement of other organs were not factors. The 10-year and 20-year survivals were 99% and 90% in stage I, 94% and 90% in stage II, 88% and 56% in stage III, 30% and 15% in stage IVa, 0% and 0% in stage IVb, 93% and 83% in the absence of involvement of the great vessels, and 54% and 20% in the presence of it. Involvement of the great vessels was also the single independent prognostic factor in the patients with stage III disease although completeness of resection or involvement of other organs were not. The 10-year and 20-year survivals in patients with stage III disease were 97% and 75% in the absence of involvement of the great vessels, and 70% and 29% in the presence of it. CONCLUSION: Although the Masaoka staging system is a valuable prognostic factor, the category of stage III is heterogeneous and consists of 2 groups with distinct prognoses depending on involvement of the great vessels.

Clinical study on lung cancer as a second primary cancer.

This study evaluates the effect of a previous cancer on the clinical characteristics and the outcome of lung cancer patients. The 313 primary lung cancer patients operated on in the Osaka University Hospital during the period 1984-1993 were reviewed. Of those, 37 had a history of previous cancer. In the lung cancer patients with a history of previous cancer, 20 had adenocarcinomas, 14 had squamous cell carcinomas, while 3 had other cancers. The previous malignancies included 13 gastric cancers, 10 head and neck cancers, 6 colorectal cancers, and 8 others. The pathological stage was 17 stage I, 1 stage II, 15 stage IIIA, 1 stage IIIB, and 3 stage IV. The 5-year survival rate was 37.9%. In patients without a history of previous cancer, there were 139 adenocarcinomas, 100 squamous cell carcinomas, and 37 others. The pathological stage was 126 stage I, 33 stage II, 74 stage IIIA, 23 stage IIIB, and 20 stage IV. The 5-year survival rate was 43.3%. There were no significant differences in the cell type, stage, or survival between the lung cancers found as the first and second cancers. Lung cancer patients with a history of previous cancer are expected to respond to a resection as well as those with lung cancer appearing as their first cancer.

Disruption of the RB pathway and cell-proliferative activity in non-small-cell lung cancers.

The pathway consisting of retinoblastoma protein (pRB), cyclin D1 and p16 (RB pathway) which is involved in the phosphorylation of pRB plays an important role in G1/S progression. The disruption of this RB pathway has been reported in several types of human neoplasm. An immunohistochemical study of 101 non-small-cell lung cancers (NSCLCs) showed loss of p16 is in 47 tumors (46.5%) and loss of pRB in 42 tumors (41.6%). In 79 of 101 NSCLCs (78.2%), the expression of p16 and pRB was complementary (p < 0.0001). Methylation of the cdkn2 gene was detected in 50% of p16-negative tumors and in 11% of p16-positive tumors. Aberrant expression of cyclin D1 was found in 45 tumors (44.5%). The cyclin-D1-positive tumors had significantly higher Ki-67 indices than the cyclin-D1-negative tumors irrespective of the tumor p16 or pRB expression. Thus, 91 (90%) of 101 NSCLCs showed disturbed expression of at least 1 of the 3 components of the RB pathway. Our results suggest that the disruption of the RB pathway plays an important role in tumorigenesis in NSCLCs and that increased cyclin-D1 expression leads to strong proliferative activity which may over-ride the suppressive effect of p16 and pRB.

p16INK4, pRB, p53 and cyclin D1 expression and hypermethylation of CDKN2 gene in thymoma and thymic carcinoma.

There have been few reports on genetic alterations in thymomas. To investigate the expression of p16INK4A, RB, p53 and cyclin D1 in thymomas, we first examined 36 thymomas (non-invasive type, 16 cases; invasive type, 20 cases) and 3 thymic carcinomas, using immunohistochemistry. Abnormal expression of p16INK4A, RB, p53 and cyclin D1 was observed in 18, 8, 10 and 7 cases, respectively. Only a subgroup of invasive thymomas and thymic carcinomas showed an inverse correlation between p16INK4A and RB expression. Subsequently, we examined the 36 thymomas and 4 thymic carcinomas for mutations in p53 and CDKN2 genes, using PCR-SSCP and direct-sequencing analyses. No mutation of these genes was detected in the thymomas and thymic carcinomas examined. A polymorphism in the 3' untranslated region of exon 3 of CDKN2 was detected in 5 cases of thymoma. We searched for hypermethylation in the promoter region of CDKN2, observing it in 4 thymomas and 1 thymic carcinoma. Our data suggest that, unlike other more common cancers, alteration of the p53 gene may not play a significant role in the tumorigenesis of thymoma. However, inactivation of p16INK4A and RB may play a role in the progression of thymoma and thymic carcinoma.

Exercise performance of rats after isogenic left or right lung transplantation followed by contralateral pulmonary artery ligation.

BACKGROUND: After single lung transplantation for pulmonary hypertension, large mismatches in ventilation/perfusion distribution persist late after transplantation. The larger graft volume of the transplanted lung after transplantation, the better the exercise performance. Because the right lung is larger in volume than the left, we compared the left single lung transplant with the right single lung transplant regarding exercise performance with an animal model. METHODS: To simulate significant ventilation/perfusion imbalance observed after single lung transplantation for pulmonary hypertension, we transplanted isogenic left or right pulmonary grafts to normal rats, and the contralateral pulmonary artery was ligated 2 weeks after transplantation. The treadmill test was performed weekly until 6 weeks after transplantation to measure maximum tolerated running speed and maximum oxygen uptake. RESULTS: Graft vital capacity of left and right pulmonary grafts were 4.5 +/- 0.43 ml (37% +/- 3.7% of recipient's predicted vital capacity) and 7.8 +/- 0.34 ml (63% +/- 2.4%), respectively (p < 0.01). Maximum tolerated speeds of left and right single lung transplants were 8 +/- 7.6 and 29 +/- 2.2 m/min, respectively, at 6 weeks after transplantation (p < 0.01). Maximal oxygen uptake values of left and right single lung transplants were 34 +/- 12.0 and 65 +/- 3.8 ml/kg/min, respectively (p < 0.01). CONCLUSIONS: Results suggest that right lung transplantation is superior to left lung transplantation for pulmonary hypertension in terms of exercise performance in this animal model.

Inversely correlated expression of p16 and Rb protein in non-small cell lung cancers: an immunohistochemical study.

Cdk4-mediated phosphorylation of Rb protein is inhibited by p16, a product of a possible tumor suppressor gene. We examined the expression of p16 and Rb protein by means of immunohistochemistry in 61 non-small cell lung cancers and have demonstrated an inverse relationship between the expression of p16 and Rb protein: 28/30 specimens that did not stain for p16 stained for Rb and 21/31 p16-positive specimens did not stain for Rb. Only 1 of the p16-negative specimens had a mutation of exon 2 of the CDKN2 gene. Our results indirectly support the theory that p16 expression is negatively regulated by the functional Rb protein.