Targeted disruption of the murine p27(Kip1) gene caused a gene dose-dependent increase in animal size without other gross morphologic abnormalities. All tissues were enlarged and contained more cells, although endocrine abnormalities were not evident. Thymic hyperplasia was associated with increased T lymphocyte proliferation, and T cells showed enhanced IL-2 responsiveness in vitro. Thus, p27 deficiency may cause a cell-autonomous defect resulting in enhanced proliferation in response to mitogens. In the spleen, the absence of p27 selectively enhanced proliferation of hematopoietic progenitor cells. p27 deletion, like deletion of the Rb gene, uniquely caused neoplastic growth of the pituitary pars intermedia, suggesting that p27 and Rb function in the same regulatory pathway. The absence of p27 also caused an ovulatory defect and female sterility. Maturation of secondary ovarian follicles into corpora lutea, which express high levels of p27, was markedly impaired.

We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% si to Flk-2/Flt-3. STK-1 is a member ofthe pe HI receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-l-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. (17,18).Cloning of STK-1. RNA was isolated by the guanidium thiocyanate method (19). First-strand cDNA, generated from 1 &g of CD34+ total RNA with priming by random hexamers and reverse transcription by Moloney murine leukemia virus reverse transcriptase (M-MLV-RT) (GIBCO/BRL), was used as template. Degenerate oligonucleotide primers (designated HHSC-PTK1 and HHSC-PTK2), corresponding to the consensus sequences of the VIb and IX subdomains of TKs, were used for PCR amplification (11). A DNA fragment (HHSC-PTK.2A) was isolated with predicted amino acid identity of 97% when compared to the murine Flk-2/Flt-3. A CD34+ cDNA library was constructed using the Superscript plasmid system (GIBCO/BRL) and screened with this fragment. Positive clones were isolated and after sequencing (20) 5' rapid amplification of cDNA ends (21,22) was used to complete the missing 5' end of the cDNA.Reverse Transcriptase PCR (RT-PCR). Equal amounts (usually 1 ,ug) of total RNA were reverse transcribed with M-MLV-RT using random hexamers or oligo(dt)15 (Boehringer Mannheim) as primers. Aliquots of this material were used with specific primer pairs for PCR amplification. Primer pairs used for STK-1 amplification included nt 91-117 and 324-304 or 878-895 and 1557-1540. Amplification consisted of 95°C for 5 min before adding Taq polymerase (New England Biolabs), followed by cycles of 95°C for 1 min, 45°C for 1 min, and 72°C for 2 min, repeated for 35 cycles. The PCR products were electrophoresed through 1% agarose gels in TAE buffer and transferred overnight to nitrocellulose (23), processed, and probed (24).

Normal expression of the hematopoietic growth factor receptor FLT3 (STK- 1@Flk2) is limited to CD34+ stem/progenitor cells. We have evaluated the expression of FLT3 by RNase protection assay and Western blotting in 161 primary bone marrow (BM) samples from patients with leukemia. FLT3 RNA was found to be expressed at a higher level than in normal BM controls in 33 of 33 B-lineage acute leukemias, 11 of 12 acute myeloid leukemias (AMLs), and 3 of 11 T-cell acute leukemias (T-ALLs). Expression of FLT3 RNA was also observed in some cases of blast crisis CML. The FLT3 signal resulted from expression on the leukemic blasts, and was not caused by increased FLT3 expression on normal CD34+ stem/progenitor cells in the leukemic samples. To determine if FLT3 protein was also overexpressed, proteins were extracted from leukemic BM samples and screened by Western blotting with anti-FLT3 antisera. FLT3 protein was not detected in normal BM controls, but was found in 14 of 14 B-lineage ALLs, 36 of 41 AMLs, and 1 of 4 T-ALLs. Stimulation of patient samples with FLT3 ligand resulted in autophosphorylation of the FLT3 receptor, suggesting the receptor is functional in these cells. These data show that FLT3 RNA and protein are aberrantly expressed by AML and ALL cells in that CD34 expression and FLT3 expression are no longer synchronous, and suggest the possibility that overexpression of FLT3 could play a role in the survival and/or proliferation of malignant clones in acute myeloid and lymphoid leukemias.