p21/Cip1/Waf1 (wild-type p53 activated fragment 1/cyclin-dependent kinase [Cdk]-interacting protein 1) is a prominent Cdk inhibitor and has been shown to be a downstream mediator of p53. In this study, we sought to clarify the clinical significance of Waf1 and the relationship between Waf1 and p53 in breast cancer. For this purpose, the expressions of Waf1 and p53 were evaluated immunohistochemically in a series of 104 patients. Waf1 was expressed in 51 (49%) of 104 tumors tested, and p53 in 33 tumors (32%). Inverse expression of these two proteins was seen in 76 cases (73%); 47 were Waf1-positive and p53-negative, and 29 were Waf1-negative and p53-positive. A comparison with clinicopathologic parameters showed that Waf1 expression correlated with negative lymph nodes (P<.01), a low histologic grade (P<.0001), and positive estrogen receptor status (P<.01). Recurrence-free survival was lower for patients with Waf1-negative tumors than for those with Waf1-positive tumors (P<.0001). In multivariate analysis, Waf1 expression and low histologic grade (1 or 2) tumors had an independent prognostic significance for recurrence-free survival. These results suggest that Waf1 is induced mainly by a p53-dependent pathway and could be a reliable indicator of recurrence in breast cancer.

The expression of the oxytocin (OT) receptor (OTR) in breast cancer was studied using newly established anti-OTR monoclonal antibodies. Immunoblotting indicated that the antibody 2F8 recognized a 70K OTR in the pregnant myometrium and breast cancer tissue. Among 57 breast cancer patients, we detected OTR immunoreactivity in 52 (91.2%) by immunohistochemistry using 2F8. Using another monoclonal antibody for different receptor domains, 1-2, the staining profile was identical in all positive samples. Of 52 OTR-positive samples, 28 were diffusely positive (> 80% of cancer cells were stained), and 24 were partially positive (< 80% cells were stained). The ratio of estrogen receptor-positive samples was slightly higher among those that were diffusely positive, but there was no apparent relationship between OTR expression and other clinical parameters. We also confirmed the expression of the OTR in positively stained samples by means of Northern blotting and RT-PCR at the transcription level. The OTR messenger RNA and RT-PCR product were the same size as those in the pregnant myometrium. We also determined the expression of the OTR using flow cytometry in four breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-361, and MDA-MB-468). However, OT had no significant effect on their growth during a short period (7 days) of culture. These findings indicated that the OTR is expressed in breast cancer derived not from the myoepithelium but from the glandular or ductal epithelium; however, the biological function of OT in breast cancer remains to be determined.

The expression of midkine (MK), a growth/differentiation factor, was assessed in 34 surgically resected specimens of primary breast cancer or mastopathy. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, all of the non-cancerous and cancerous tissues were found to express MK except for one breast cancer specimen. Northern blot analysis revealed that MK mRNA was also expressed in the normal breast tissues examined. Immunohistochemical analysis of the MK protein was performed on a limited number of the specimens, showing that some cancerous tissues were immunoreactive with anti-MK antibodies. Furthermore, using RT-PCR analysis, expression of not only the wild-type but also a truncated form of MK, which was recently found in various human tumor cell lines, was detected in 6 of 26 cancerous tissues but not in non-cancerous tissues.

Summary p21 (WAF1/CIP1) protein expression in various thyroid tissues, including thyroid carcinoma, was studied by means of immunohistochemistry using anti-p21 monoclonal antibody. Normal follicles and hyperplasias rarely expressed p21, whereas immunohistochemically positive cells were also too rarely found in follicular adenomas to justify these cases being classified as positive. Twenty eight of the 93 carcinomas examined (30.1%), however, were positive for p21. Of the p21-positive cases, 80% of the undifferentiated and 28.6% of the poorly differentiated carcinomas showed lesions co-expressing p21 and p53. If diffuse immunoreactivity of p53 reflects the p53 mutation, our results indicate that p21 in these carcinomas can be induced by p53-independent as well as by p53-dependent pathways. On the other hand, well-differentiated carcinomas did not co-express these two proteins and it therefore remains unclear whether p53-independent or p53-dependent pathways are predominant in this type of carcinoma. The incidence of expression of p21 was very similar in undifferentiated (26.3%), poorly (28.0%) and well-differentiated carcinomas (32.7%), even though they are characterised by different degrees of malignancy. Furthermore, no correlation between p21 expression and either clinical parameters or patient's prognosis could be established. These results suggest that p21 is only marginally related to the characteristics of thyroid carcinoma and can play only an adjuvant role in regulating the progression of this carcinoma.

The immunolocalization of the pl6 and cdk4 proteins was investigated in 65 retinoblastoma gene product (pRB)-positive and 20 pRB-negative breast carcinomas. These proteins were expressed in similar lesions in 84.6% of the pRB-positive and 100% of the pRB-negative carcinomas. Diffuse expression of pl6 was observed in 73.8 and 70.0% of the pRB-positive and -negative cases, respectively. cdk4 and p 16 expression was significantly more heterogeneous in tumors of larger sizes and/or at higher stages. These findings suggest that p 16 can be induced regardless of pRB status and Rb gene function in primary breast carcinoma and that it modulates the cell cycle progression in association with cdk4.